Variants of chymosin with improved properties

ABSTRACT

Variants of chymosin with improved properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. National Stage of International Application PCT/EP2016/064414, filed Jun. 22, 2016, and claims priority to European Patent Application No, 15173099.1, filed Jun. 22, 2015.

FIELD OF THE INVENTION

The present invention relates to variants of chymosin with improved properties.

BACKGROUND ART

Chymosin (EC 3.4.23.4) and pepsin (EC 3.4.23.1), the milk clotting enzymes of the mammalian stomach, are aspartic proteases belonging to a broad class of peptidases.

When produced in the gastric mucosal cells, chymosin and pepsin occur as enzymatically inactive pre-prochymosin and pre-pepsinogen, respectively. When chymosin is excreted, an N-terminal peptide fragment, the pre-fragment (signal peptide) is cleaved off to give prochymosin including a pro-fragment. Prochymosin is a substantially inactive form of the enzyme which, however, becomes activated under acidic conditions to the active chymosin by autocatalytic removal of the pro-fragment. This activation occurs in vivo in the gastric lumen under appropriate pH conditions or in vitro under acidic conditions.

The structural and functional characteristics of bovine, i.e. Bos taurus, pre-prochymosin, prochymosin and chymosin have been studied extensively. The pre-part of the bovine pre-prochymosin molecule comprises 16 aa residues and the pro-part of the corresponding prochymosin has a length of 42 aa residues. The active bovine chymosin comprises 323 aa.

Chymosin is produced naturally in mammalian species such as bovines, camels, caprines, buffaloes, sheep, pigs, humans, monkeys and rats.

Bovine and camel chymosin has for a number of years been commercially available to the dairy industry.

Enzymatic coagulation of milk by milk-clotting enzymes, such as chymosin and pepsin, is one of the most important processes in the manufacture of cheeses. Enzymatic milk coagulation is a two-phase process: a first phase where a proteolytic enzyme, chymosin or pepsin, attacks K-casein, resulting in a metastable state of the casein micelle structure and a second phase, where the milk subsequently coagulates and forms a coagulum (reference 1). Besides facilitating coagulation of milk by cleaving κ-casein, chymosins cleave β-casein (β-casein), primarily between Leu192 and Tyr193, resulting in the formation of a β(193-209) peptide. Further proteolysis of β(193-209) and formation of short hydrophobic peptides may result in an undesirable bitter flavor of the product.

-   WO02/36752A2 (Chr. Hansen) describes recombinant production of camel     chymosin. -   WO2013/174840A1 (Chr. Hansen) describes mutants/variants of bovine     and camel chymosin. -   WO2013/164479A2 (DSM) describes mutants of bovine chymosin.

The references listed immediately below may in the present context be seen as references describing mutants of chymosin:

-   Suzuki et al: Site directed mutagenesis reveals functional     contribution of Thr218, Lys220 and Asp304 in chymosin, Protein     Engineering, vol. 4, January 1990, pages 69-71; -   Suzuki et al: Alteration of catalytic properties of chymosin by     site-directed mutagenesis, Protein Engineering, vol. 2, May 1989,     pages 563-569; -   van den Brink et al: Increased production of chymosin by     glycosylation, Journal of biotechnology, vol. 125, September 2006,     pages 304-310; -   Pitts et al: Expression and characterisation of chymosin pH optima     mutants produced in Tricoderma reesei, Journal of biotechnology,     vol. 28, March 1993, pages 69-83; -   M. G. Williams et al: Mutagenesis, biochemical characterization and     X-ray structural analysis of point mutants of bovine chymosin,     Protein engineering design and selection, vol. 10, September 1997,     pages 991-997; -   Strop et al: Engineering enzyme subsite specificity: preparation,     kinetic characterization, and x-ray analysis at 2.0 ANG resolution     of Val111phe site mutated calf chymosin, Biochemistry, vol. 29,     October 1990, pages 9863-9871; -   Chitpinityol et al: Site-specific mutations of calf chymosin B which     influence milk-clotting activity, Food Chemistry, vol. 62, June     1998, pages 133-139; -   Zhang et al: Functional implications of disulfide bond, Cys45-Cys50,     in recombinant prochymosin, Biochimica et biophysica acta, vol.     1343, December 1997, pages 278-286.

None of the prior art references mentioned above describe directly and unambiguously any of the chymosin variants with lowered β-casein cleavage frequency at similar clotting activity compared to the parent from which the variant is derived, as described below.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide variants of chymosin which, when compared to the parent polypeptide, has a lower lowered βcasein cleavage frequency while substantially maintaining its clotting efficiency.

Accordingly, the present invention provides isolated chymosin polypeptide variants characterized in that:

-   -   (a) the isolated chymosin polypeptide variant has a specific         clotting ac-tivity (IMCU/mg total protein) that is at least 80%         of the specific clotting activity of isolated camel chymosin         polypeptide characterized by SEQ ID NO:4; and     -   (b) the isolated chymosin polypeptide variant cleaves β-casein         with a frequency of less than 50% of the frequency of β-casein         cleavage of isolated camel chymosin polypeptide characterized by         SEQ ID NO:4, wherein β-casein cleavage is determined by         quantifying β-casein peptides obtained by incubating skim milk         with the chymosin variant or the camel chymosin, wherein         quantification is carried out by RP-HPLC coupled to an ESI-Q-TOF         mass spectrometer.

The isolated chymosin polypeptide variant of present invention may be derived from a parent polypeptide has at least 80%, such as at least e.g. 85%, 95%, 97%, 98%, 99%, 100% sequence identity with the polypeptide of SEQ ID NO:4 (camel chymosin).

In a related aspect, the isolated chymosin polypeptide variant of present invention has at least 70%, such as at least e.g. 75%, 80%, 90%, 100%, 110%, 120%, 130% or 150% of the specific clotting activity of isolated camel chymosin polypeptide characterized by SEQ ID NO:4.

In yet a related aspect, the isolated chymosin polypeptide variant of present invention preferably has at least has less than 50%, such as e.g. less than 40%, less than 30%, less than 20%, less than 15%, less than 10% or less than 6% of the unspecific proteolytic activity (P) of isolated camel chymosin polypeptide characterized by SEQ ID NO:4.

In a further related aspect, the isolated chymosin polypeptide variant of present invention has at least has a C/P ratio of at least 300%, 400%, 500%, 600%, 700%, 800%, 1000%, 1200%, 1400% or 1600% of the C/P ratio of isolated camel chymosin polypeptide characterized by SEQ ID NO:4.

The isolated chymosin polypeptide variant of present invention may comprise one or more amino acid substitutions, deletions or insertions, wherein the one or more substitution, deletion or insertion is specified in relation to the amino acid sequence of SEQ ID NO:4: Y11, L130, S132, V32, S226, R266, L12, V221, S255, S277, L222, L253, M157, V260, S271, H76, K19, V183, S164, I263, V51, T239, Y307, R67, G251, R61, Q288, E83, D59, V309, S273, G251, S154, Y21, V203, L180, E294, G289, L215, D144, I303, L105, T284, Y127, V248, K321, V205, E262, K231, R316, M256, D158, D59, N249, L166, R242 or I96, and more specifically such as e.g. Y11I, Y11V, L130I, S132A, V32L, S226T, R266V, L12M, V221M, S255Y, S277N, L222I, L253I, M157L, V260T, S271P, H76Q, K19T, V183I, S164G, I263L, V51L, T239S, Y307F, R67Q, G251D, R61Q, Q288E, E83S, D59N, V309I, S273Y, G251W, S154A, Y21S, V203A, L180I, E294Q, G289S, L215V, D144Q, I303L, L105E, T284S, Y127F, V248I, K321P, V205I, E262T, K231N, R316L, M256L, D158S, D59N, N249E, L166V, R242E and/or I96L.

The present invention further provides methods of making the isolated chymosin polypeptide variants of present invention, methods of making a food or feed product using the isolated chymosin polypeptide variants, food and feed products comprising these variants as well as the use of the variants for making food and feed products.

In a related alternative aspect, the invention relates to methods for making an isolated chymosin polypeptide with decreased comprising the following steps:

-   -   (a): making an alteration at one or more positions in the DNA         sequence encoding the polypeptide of SEQ ID NO:4, wherein the         alteration comprises a substitution, a deletion or an insertion         in at least one amino acid position corresponding to any of         positions:

Y11, L130, S132, V32, S226, R266, L12, V221, S255, S277, L222, L253, M157, V260, S271, H76, K19, V183, S164, I263, V51, T239, Y307, R67, G251, R61, Q288, E83, D59, V309, S273, G251, S154, Y21, V203, L180, E294, G289, L215, D144, I303, L105, T284, Y127, V248, K321, V205, E262, K231, R316, M256, D158, D59, N249, L166, R242 or I96 in SEQ ID NO:4;

-   -   (b): producing and isolating the altered polypeptide of step         (a).

The isolated chymosin produced by the methods above, may comprise one or more of the following substitutions:

Y11I, Y11V, L130I, S132A, V32L, S226T, R266V, L12M, V221M, S255Y, S277N, L222I, L253I, M157L, V260T, S271P, H76Q, K19T, V183I, S164G, I263L, V51L, T239S, Y307F, R67Q, G251D, R61Q, Q288E, E83S, D59N, V309I, S273Y, G251W, S154A, Y21S, V203A, L180I, E294Q, G289S, L215V, D144Q, I303L, L105E, T284S, Y127F, V248I, K321P, V205I, E262T, K231N, R316L, M256L, D158S, D59N, N249E, L166V, R242E and/or I96L.

In a related aspect the isolated chymosin polypeptide variant of present invention and the variant produced by the methods above may comprise a combination of substitutions and wherein each substitution is specified in relation to the amino acid sequence of SEQ ID NO:4:

I96+G163+V221; R67+H76+S132+V248+S271; R67+L130+M157;

V136+V221+L222+S226; S132+R254+V259+Y307; V32+I96+S277;

L130+M142+I200+V259+E294; L130+S132+V32;

L130+G163+Y307; R61+L166+T239; L130+T239+S277+L295;

D98+H146+V203+I263+S271; S132+V221+S255+S273+V317;

H76+L222+G251; H76+K231+G244;

Y127+S132+D158; V221+V248+L253+L295; V32+R61+H146;

V32+E294+R316+V317; H76+I96+D158; D98+M157+V183;

S226+G244+I263+G289; G70+L130+Y268; D59+V248+L222+V248;

R67+G70+H146+Q188+S226; 574+H76+M142+M157+G163;

R61+S226+T239+V248+G251; V32+L130+R145+L222+D279;

D59+L222+G251+E83+Q162; D59+L222+G251+F17+Y21;

D59+L222+G251+H76+S164; D59+L222+G251+K62+M165; D59+L222+G251+Q162+V155; D59+L222+G251+S273+L166; D59+L222+G251+Y268+V198; D59+L222+G251+S273+F66; D59+L222+G251+M165+L166; D59+L222+G251+H76+M165; D59+L222+G251+F17+S273; D59+L222+G251+L166+I45;

D59+L222+G251+L180+T284; D59+L222+G251+V32+L12+T284;

D59+L222+G251+Y21+L166; D59+L222+G251+V155+E262+V32;

D59+L222+G251+L105+S164; D59+L222+G251+Y21+L215+L105;

D59+L222+G251+I96+T177+K321; D59+L222+G251+F17+T284+V203;

D59+L222+G251+V32+K321+V260; D59+L222+G251+V198+V32+E83;

D59+L222+G251+I96+V203+V309; D59+L222+G251+Y268+L215+V32;

D59+L222+G251+H76+L105+V260; D59+L222+G251+Y21+H76+Y268;

D59+L222+G251+S164+R266+I96; D59+L222+G251+H181+F66+V32;

D59+L222+G251+H181+R266+D267; D59+L222+G251+Y268+L12+D267;

D59+L222+G251+L166+E262+T177; D59+L222+G251+F66+Q288+I96;

D59+L222+G251+V203+R266+F223; D59+L222+G251+1303+S154+V260;

D59+L222+G251+Y21+T284+I96; D59+L222+G251+Q288+K19+T177;

D59+L222+G251+K62+Y268+K19;

L12+Y21+D59+H76+M165+V198+L222+G251+Q288;

L12+Y21+D59+H76+M165+L222+G251+S273;

L12+D59+H76+M165+V198+L222+G251+S273+K321;

L12+D59+H76+S154+M165+V203+L222+G251+V309;

L12+D59+H76Q+D98+L222;

L12+K19+V32+D59+H76+D144+M165+L222+G251;

L12+Y21+D59+H76+M165+V203+L222+G251+E262;

L12+V51+H76+M165+G251;

L12+D59+F66+H76+M165+L180+L222+G251+V309;

L12+D59+H76+S154+M165+L222+G251+Q288;

L12+D59+H76+D98+M165+L222+G251+E262+Q288;

L12+V51+D59+H76+L166+L222+G251;

L12+D59+H76+D144+M165+V203+L222;

L12+D59+144+M165+L166+L222+G251;

L12+K19+D59+H76+S154+M165+V198+L222+G251;

L12+H76+D98+M165+L222+G251;

L12+V32+D59+H76+M165+L180+V198+L222+G251;

L12+D59+H76+S154+M165+S273;

L12+V51+D59+F66Y+H76Q+M165E+V203A+L222I+G251W;

L12+V32+H76+M165+L222+E262; L12+N50+D59+H76+M165+G251+E262;

V51+D59+H76+M165+L180+L222+G251+E262;

L12+D59+H76+M165+G251+Q288+V309+K321;

L12+N50+D59+V203+L222+G251; L12+D59+H76+L180+L222+G251+K321;

L12+Y21+D59+M165+L222+K321; D59+H76+M165+L166+V198+L222;

L12+K19+N50+D59+H76+M165+L222+Q288;

L12+Y21+N50+D59+F66+H76+D144+M165+L222+G251;

H76+S132+S164+L222+N249+G251;

Y21+D59+H76+S164+L166+N249+G251+S273;

D59+H76+S164+L222+R242+S273+V309;

D59+H76+L130+L166+L222+N249+G251+S273;

Y21+D59+S164+L222+R242+G251+S273+V309;

K19+Y21+D59+H76+S132+S164+L222+G251+S273;

D59+H76+I96+L130+S164+L222+R242+G251;

H76+S164+L166+L222+S226+S273; K19+D59+I96+S164+L222+G251;

Y21+H76+S164+L222+R242+G251+S273;

H76+I96+S164+L222+R242+G251+S273;

H76+S164+L222+N249+G251+S273+V309;

K19+D59+H76+S164+L222+N249+S273;

Y21+D59+H76+S164+L222+S226+G251+S273+V309;

H76+S164+L166+L222+R242+G251+S273;

D59+H76+I96+S164+L222+S226+N249+G251+S273;

D59+H76+L130+S164+L166+L222+G251+S273+V309;

D59+S132+S164+L222+R242+N249+G251+S273;

H76+I96+S164+G251+S273+V309; D59+H76+L130+S164+G251+V309;

K19+D59+S164+L166+L222+S226+G251+S273;

D59+H76+I96+S132+S164+L222+S226+G251+S273;

K19+D59+H76+I96+S164+L166+L222+G251+S273;

K19+D59+H76+L130+S164+L222+S226+G251+S273;

K19+D59+H76+S132+L222+G251+S273+V309;

H76+L130+L222+S226+G251+S273;

K19+Y21+D59+H76+L130+S164+L222+S273;

Y21+D59+H76+I96+S164+L222+N249+G251+S273;

K19+D59+H76+S164+R242+N249+G251+S273;

D59+H76+S164+L222+S226+R242;

D59+H76+I96+S132+S164+L166+L222+G251+S273;

D59+H76+S132+S164+L166+S273;

Y21+D59+S164+L222+S226+N249+G251+S273;

D59+H76+L130+S132+S164+L222+R242+G251+S273;

D59+H76+S164+L166+L222+N249+G251+S273+V309;

D59+H76+I96+S164+L222+S226+G251+S273+V309;

K19+D59+H76+L166+L222+R242+G251+S273;

Y21+D59+H76+I96+L222+S273;

D59+H76+I96+L130+S164+L222+N249+G251+S273;

L130+S164+L222+S273; K19+Y21+H76+S164+L222+G251+S273;

Y21+D59+H76+L130+S132+S164+L222+G251+S273;

D59+H76+S226+R242+G251+S273; K19+D59+I96+S164+L222+G251;

Y11+K19+D59+I96+L222+R242+G251; K19+D59+I96+S164+G251;

K19+I96+S164+L166+L222+R242;

K19+D59+I96+S164+L166+L222+R242+G251+L253;

D59+196+S164+L222+R242+L253+I263;

K19+D59+E83+196+L222+G251+I263;

Y11+K19+D59+S164+L222+G251+I263;

K19+D59+I96+S164+L166+G251+L253;

K19+I96+S164+L222+N249+G251+L253; K19+I96+L222+R242+L253;

K19+E83+I96+S164+L222+R242+G251+L253;

D59+E83+I96+S164+L222+G251;

K19+D59+I96+S164+L222+R242+N249+G251;

K19+I96+S164+L166+L222+N249+I263; D59+I96+L166+L222+R242+G251;

K19+D59+E83+S164+L166+L222+R242+G251;

Y11+K19+D59+E83+I96+S164+L222+N249;

K19+E83+I96+S164+L222+R242+L253;

K19+D59+I96+S164+L166+L222+R242+N249;

Y11+K19+D59+I96+S164+L166+L222+R242+G251+L253;

K19+I96+S164+L222+R242+I263; Y11+D59+I96+S164+L222+G251+L253;

K19+D59+196+S164+L166+L222+R242+I263;

Y11+K19+D59+I96+S164+L166+L222+G251;

K19+196+S164+L166+L222+R242+N249+G251+I263;

K19+I96+S164+R242+L253; K19+D59+E83+I96+S164+L222+G251;

K19+D59+I96+S164+L222+N249+G251+I263;

K19+D59+I96+S164+L222+N249+G251+L253+I263;

Y11+K19+I96+S164+L222+R242+G251;

I96+S164+L222+R242+N249+G251+1263;

K19+D59+196+S164+L166+L222+R242+G251+1263;

K19+D59+I96+S164+L222+R242+N249+L253;

H76+I96+S164+L222+R242+G251+S273;

K19+E83+I96+S164+L222+R242+N249+G251+L253;

I96+S164+L166+L222+R242+N249+1263;

Y11+K19+E83+I96+S164+L166+L222+R242+G251;

Y11+K19+I96+S164+L166+L222+R242;

Y11+E83+I96+S164+L222+R242+G251+L253+I263;

Y11+I96+S164+L222+R242+N249+L253+1263;

K19+196+S164+L166+L222+R242+N249+I263;

Y11+E83+I96+S164+L222+R242+L253+I263;

K19+E83+I96+S164+L166+L222+R242+N249+G251+L253;

I96+S164+L222+R242+G251+S274; H76+I96+S164+L222+R242+G251;

I96+S164+L222+R242+G251; V32+N100+N291; V221+N100+N291;

D290+N100+N291; V136+N100+N291; E240+N100+N291; R242+N100+N291;

G289+N100+N291; N292+N100+N291; L295+N100+N291; V136+N100+N291;

D290+N100+N291; F119+N100+N291; Q280+N100+N291; F282+N100+N291;

R254+N100+N291; R242+N100+N291; V203+N100+N291; N249+N100+N291;

H56+N100+N291; 574+N100+N291; A131+N100+N291; Y190+N100+N291;

I297+N100+N291; H76+N100+N291; S273+N100+N291; K19+N100+N291;

D59+N100+N291; L222+N100+N291; V309+N100+N291; I96+N100+N291;

Y21+N100+N291; L130+N100+N291; S132+N100+N291; S226+N100+N291;

G251+N100+N291; Y243+N100+N291; S273+N100+N291;

R242+Q280+N100+N291; R242+N252+N100+N291;

N252+Q280+N100+N291; Y243+Q280+N100+N291; Y243+N252+N100+N291;

R254+Q280+N100+N291; S273+Q280+N100+N291;

R242+G251+N100+N291; R242+G251+Q280+N100+N291;

R242+S273+Q280+N100+N291; N252+S273+Q280+N100+N291;

G251+S273+Q280+N100+N291; R242+R254+Q280+N100+N291;

R242+R254+S273+Q280+N100+N291; Y243+R254+S273+N100+N291;

V223+N252+N291; E290+N252+N291; A117+N252+N291; I136+N252+N291;

Q242+N252+N291; Q278+N252+N291; S289+N252+N291;

Q294+N252+N291; D249+N252+N291; D251+N252+N291;

G244+N252+N291; Q56+N252+N291; L32+N252+N291; K71+N252+N291;

P72+N252+N291; Q83+N252+N291; V113+N252+N291; E133+N252+N291;

Y134+N252+N291; K71+N252+N291; Y11+N100+N291;

Y11+D290+N100+N291; L12+N100+N291; D13+N100+N291;

D13+N100+N291; R67+N100+L130+M157+V248+N291;

N100+L130+S132+M157+K231; R67+196+L130+M157+L222+M256;

R67+L130+S132+M157+R242+V248; R67+N100+M157+R242+M256;

R67+G70+M157+R242+V248; V32+R67+M157+L222+R242;

Y11+R67+M157+V248+M256; R67+V136+M157+L222+V248;

L130+M157+V248+M256+N291; R67+196+L130+M157+K231+R242;

V32+R67+L130+M157+L222+K231; L130+V136+M157+L222+N292;

R67+G70+M157+L222+N291; V32+R67+L130+K231+N292;

Y11+R67+N100+L130+V136+M157; R67+L130+L222+R242+M256;

R67+M157+L222+V248+N292; V32+R67+M157+M256+N291;

R67+L130+S132+M157+L222+N292; R67+N100+L130+M157+K231+N291;

R67+L130+K231+V248+N291; Y11+R67+L130+M157+L222+K231;

I45+L130+M157+K231+R242; V32+R67+V136+M157+N291;

R67+N100+L130+D158+V248; I45+R67+L130+M157+L222+K231;

V32+R67+L130+S132+M157+V248; Y11+R67+L130+M157+N291+N292;

R67+N100+L130+M157+L222+K231; 145+R67+G70+L130+S132;

I45+R67+L130+V248+N292; Y11+R67+L130+M157+L222+R242;

R67+N100+D158+L130+M157+L222; R67+L130+V136+M157+K231+V248;

I45+R67+L130+L222+N291; R67+G70+L130+M157+K231+M256;

V32+R67+L130+M157+D158+V248; R67+L130+M157+D158+R242+N291;

R67+L130+M157+D158+K231+N292; R67+L130+V248+M256+N292;

V32+R67+I96+L130+M157+V248; R67+I96+N100+L130+M157+N292;

V32+R67+G70+N100+M157; V32+R67+L130+M157+K231+M256;

R67+I96+M157+L222+K231; R67+M157+L222+K231+V248;

R67+L130+M157+R242+M256+N292; R67+L222+K231+V248;

R67+S132+L222+K231+R242+V248;

Y11+K19+D59+S164+L166+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+G251;

Y11+K19+D59+I96+L166+L222+R242+N249+G251+L253;

Y11+K19+D59+I96+S164+L166+R242;

Y11+K19+D59+I96+S164+L222+R242+G251;

Y11+K19+D59+I96+S164+L166+R242+N249+G251+L253;

Y11+K19+D59+I96+S164+L166+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+N249+G251+L253;

Y11+K19+D59+L166+L222+R242+N249+G251+L253;

Y11+K19+D59+I96+S164+L166+L222+R242+N249;

Y11+K19+D59+S164+L166+L222+R242+G251;

Y11+K19+D59+I96+S164+R242+G251;

Y11+D59+I96+S164+L166+L222+R242+G251+L253;

Y11+D59+I96+S164+L166+L222+R242+G251;

Y11+D59+I96+S164+L166+L222+R242+G251+L253;

Y11+K19+D59+I96+S164+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+N249+L253;

Y11+K19+D59+I96+S164+L166+L222+R242+N249+G251;

Y11+K19+I96+S164+L166+R242+N249+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+G251;

Y11+K19+D59+I96+S164+L222+R242+N249+G251;

Y11+K19+L222+R242+N249+G251; Y11+K19+I96+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+N249+G251;

Y11+K19+I96+S164+L166+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L166+L222+R242+N249+G251;

Y11+I96+S164+L166+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L222+R242+N249;

Y11+K19+D59+I96+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L222+R242;

Y11+K19+D59+I96+S164+L166+R242+G251;

Y11+K19+D59+S164+L166+L222+R242+G251;

Y11+I96+L222+R242+N249+G251; Y11+I96+S164+L222+R242;

Y11+K19+I96+L166+L222+R242+G251;

Y11+D59+I96+S164+L222+R242+G251;

Y11+D59+I96+S164+L222+R242+N249+G251;

Y11+K19+D59+I96+S164+L222+R242+N249+G251;

Y11+D59+I96+S164+L166+L222+R242+G251;

Y11+K19+D59+I96+L222+R242+G251;

Y11+K19+S164+L166+L222+R242+N249+G251;

Y11+D59+I96+S164+L166+L222+R242+N249+G251, such as e.g.:

I96L+G163E+V221M;

R67Q+H76Q+S132A+V248I+S271P;

R67Q+L130I+M157L;

V136I+V221M+L222I+S226T;

S132A+R254S+V259I+Y307F;

V32L+I96L+S277N;

L130I+M142I+I200V+V259I+E294Q;

L130I+G163E+Y307F;

R61S+L166V+T239S;

L130I+T239S+S277N+L295K;

L130I+S132A+V32L;

D98V+H146R+V203A+I263L+S271P;

S132A+V221M+S255Y+S273Y+V317L;

H76Q+L222I+G251W;

H76Q+K231N+G244D;

Y127F+S132A+D158S;

V221M+V248I+L253I+L295K;

V32L+R61Q+H146R;

V32L+E294Q+R316L+V317L;

H76Q+I96L+D158S;

D98V+M157L+V183I;

S226T+G244D+I263L+G289S;

G70D+L130I+Y268F;

D59N+V248I+L222I+V248I;

R67Q+G70N+H146R+Q188E+S226T;

S74F+H76Q+M142I+M157L+G163E;

R61Q+S226T+T239S+V248I+G251W;

V32L+L130I+R145Q+L222I+D279E;

D59N+L222I+G251D+E83S+Q162S;

D59N+L222I+G251W+F17Y+Y21S;

D59N+L222I+G251D+H76Q+S164G;

D59N+L222I+G251D+K62Q+M165E;

D59N+L222I+G251D+Q162S+V155F;

D59N+L222I+G251D+S273Y+L166V;

D59N+L222I+G251D+Y268F+V198I;

D59N+L222I+G251D+S273Y+F66Y;

D59N+L222I+G251D+M165E+L166V;

D59N+L222I+G251D+H76Q+M165E;

D59N+L222I+G251D+F17Y+S273Y;

D59N+L222I+G251D+L166V+145V;

D59N+L222I+G251W+L180I+T284S;

D59N+L222I+G251D+V32L+L12M+T284S;

D59N+L222I+G251D+Y21S+L166V;

D59N+L222I+G251D+V155F+E262T+V32L;

D59N+L222I+G251D+L105E+S164G;

D59N+L222I+G251D+Y21S+L215V+L105E;

D59N+L222I+G251D+196L+T177S+K321P;

D59N+L222I+G251D+F17Y+T284S+V203A;

D59N+L222I+G251D+V32L+K321P+V260T;

D59N+L222I+G251D+V198I+V32L+E83S;

D59N+L222I+G251D+196L+V203A+V309I;

D59N+L222I+G251D+Y268F+L215V+V32L;

D59N+L222I+G251D+H76Q+L105E+V260T;

D59N+L222I+G251D+Y21S+H76Q+Y268F;

D59N+L222I+G251D+S164G+R266V+196L;

D59N+L222I+G251D+H181N+F66Y+V32L;

D59N+L222I+G251D+H181N+R2661+D267Q;

D59N+L222I+G251D+Y268F+L12M+D267Q;

D59N+L222I+G251D+L166V+E262T+T177S;

D59N+L222I+G251D+F66Y+Q288E+196L;

D59N+L222I+G251D+V203A+R266V+F223A;

D59N+L222I+G251D+1303L+S154A+V260T;

D59N+L222I+G251D+Y21S+T284S+196L;

D59N+L222I+G251D+Q288E+K19T+T177S;

D59N+L222I+G251D+K62Q+Y268F+K19T

L12M+Y21S+D59N+H76Q+M165E+V198I+L222I+G251D+Q288E;

L12M+Y21S+D59N+H76Q+M165E+L222I+G251W+S273Y;

L12M+D59N+H76Q+M165E+V198I+L222I+G251D+S273Y+K321P;

L12M+D59N+H76Q+S154A+M165E+V203A+L222I+G251D+V309I;

L12M+D59N+H76Q+D98V+L222I;

L12M+K19T+V32L+D59N+H76Q+D144Q+M165E+L222I+G251D;

L12M+Y21S+D59N+H76Q+M165E+V203A+L222I+G251D+E262T;

L12M+V51L+H76Q+M165E+G251D;

L12M+D59N+F66Y+H76Q+M165E+L180I+L222I+G251D+V309I;

L12M+D59N+H76Q+S154A+M165E+L222I+G251W+Q288E;

L12M+D59N+H76Q+D98V+M165E+L222I+G251D+E262T+Q288E;

L12M+V51L+D59N+H76Q+L166V+L222I+G251D;

L12M+D59N+H76Q+D144Q+M165E+V203A+L222I;

L12M+D59N+144Q+M165E+L166V+L222I+G251D;

L12M+K19T+D59N+H76Q+S154A+M165E+V198I+L222I+G251D;

L12M+H76Q+D98V+M165E+L222I+G251W;

L12M+V32L+D59N+H76Q+M165E+L180I+V198I+L222I+G251D;

L12M+D59N+H76Q+S154A+M165E+S273Y;

L12M+V51L+D59N+F66Y+H76Q+M165E+V203A+L222I+G251W;

L12M+V32L+H76Q+M165E+L222I+E262T;

L12M+N50D+D59N+H76Q+M165E+G251W+E262T;

V51L+D59N+H76Q+M165E+L180I+L222I+G251D+E262T;

L12M+D59N+H76Q+M165E+G251D+Q288E+V309I+K321P;

L12M+N50D+D59N+V203A+L222I+G251D;

L12M+D59N+H76Q+L180I+L222I+G251W+K321P;

L12M+Y21S+D59N+M165E+L222I+K321P;

D59N+H76Q+M165E+L166V+V198I+L222I;

L12M+K19T+N50D+D59N+H76Q+M165E+L222I+Q288E;

L12M+Y21S+N50D+D59N+F66Y+H76Q+D144Q+M165E+L222I+G251D;

H76Q+S132A+S164G+L222I+N249D+G251D;

Y21S+D59N+H76Q+S164G+L166V+N249D+G251D+S273Y;

D59N+H76Q+S164G+L222I+R242E+S273Y+V309I;

D59N+H76Q+L130I+L166V+L222I+N249D+G251D+S273Y;

Y21S+D59N+S164G+L222I+R242E+G251D+S273Y+V309I;

K19T+Y21S+D59N+H76Q+S132A+S164G+L222I+G251D+S273Y;

D59N+H76Q+196L+L130I+S164G+L222I+R242E+G251D;

H76Q+S164G+L166V+L222I+S226T+S273Y;

K19T+D59N+I96L+S164G+L222I+G251D;

Y21S+H76Q+S164G+L222I+R242E+G251D+S273Y;

H76Q+196L+S164G+L222I+R242E+G251D+S273Y;

H76Q+S164G+L222I+N249D+G251D+S273Y+V309I;

K19T+D59N+H76Q+S164G+L222I+N249D+S273Y;

Y21S+D59N+H76Q+S164G+L222I+S226T+G251D+S273Y+V309I;

H76Q+S164G+L166V+L222I+R242E+G251D+S273Y;

D59N+H76Q+196L+S164G+L222I+S226T+N249D+G251D+S273Y;

D59N+H76Q+L130I+S164G+L166V+L222I+G251D+S273Y+V309I;

D59N+S132A+S164G+L222I+R242E+N249D+G251D+S273Y;

H76Q+196L+S164G+G251D+S273Y+V309I;

D59N+H76Q+L130I+S164G+G251D+V309I;

K19T+D59N+S164G+L166V+L222I+S226T+G251D+S273Y;

D59N+H76Q+196L+S132A+S164G+L222I+S226T+G251D+S273Y;

K19T+D59N+H76Q+196L+S164G+L166V+L222I+G251D+S273Y;

K19T+D59N+H76Q+L130I+S164G+L222I+S226T+G251D+S273Y;

K19T+D59N+H76Q+S132A+L222I+G251D+S273Y+V309I;

H76Q+L130I+L222I+S226T+G251D+S273Y;

K19T+Y21S+D59N+H76Q+L130I+S164G+L222I+S273Y;

Y21S+D59N+H76Q+196L+S164G+L222I+N249D+G251D+S273Y;

K19T+D59N+H76Q+S164G+R242E+N249D+G251D+S273Y;

D59N+H76Q+S164G+L222I+S226T+R242E;

D59N+H76Q+196L+S132A+S164G+L166V+L222I+G251D+S273Y;

D59N+H76Q+S132A+S164G+L166V+S273Y;

Y21S+D59N+S164G+L222I+S226T+N249D+G251D+S273Y;

D59N+H76Q+L130I+S132A+S164G+L222I+R242E+G251D+S273Y;

D59N+H76Q+S164G+L166V+L222I+N249D+G251D+S273Y+V309I;

D59N+H76Q+196L+S164G+L222I+S226T+G251D+S273Y+V309I;

K19T+D59N+G251D+S273; H76Q+L166V+L222I+R242E+G251D+S273Y;

Y21S+D59N+H76Q+196L+L222I+S273Y;

D59N+H76Q+I96L+L130I+S164G+L222I+N249D+G251D+S273Y;

L130I+S164G+L222I+S273Y;

K19T+Y21S+H76Q+S164G+L222I+G251D+S273Y;

Y21S+D59N+H76Q+L130I+S132A+S164G+L222I+G251D+S273Y;

D59N+H76Q+S226T+R242E+G251D+S273Y;

K19T+D59N+I96L+S164G+L222I+G251D;

Y11I+K19T+D59N+196V+L222I+R242D+G251D;

K19S+D59N+I96V+S164G+G251D; K19S+I96L+S164G+L166V+L222I+R242E;

K19T+D59N+I96L+S164G+L166V+L222I+R242D+G251D+L253I;

D59N+I96L+S164G+L222I+R242E+L253I+I263L;

K19T+D59N+E83T+I96L+L222I+G251D+I263L;

Y11I+K19T+D59N+S164G+L222I+G251D+I263V;

K19T+D59N+I96L+S164G+L166I+G251D+L253V;

K19T+196V+S164G+L222I+N249D+G251D+L253I;

K19T+196L+L222I+R242E+L253I;

K19T+E83S+196L+S164G+L222I+R242E+G251D+L253I;

D59N+E83T+I96L+S164N+L222V+G251D;

K19S+D59N+I96L+S164G+L222I+R242E+N249E+G251D;

K19T+I96L+S164G+L166V+L222I+N249D+I263L;

D59N+I96L+L166V+L222I+R242E+G251D;

K19T+D59N+E83T+S164G+L166V+L222I+R242D+G251D;

Y11I+K19T+D59N+E83S+I96L+S164G+L222I+N249D;

K19T+E83T+I96L+S164G+L222I+R242E+L253V;

K19T+D59N+I96L+S164G+L166I+L222I+R242E+N249D;

Y11V+K19T+D59N+I96L+S164G+L166V+L222I+R242E+G251D+L253I;

K19T+I96L+S164N+L222I+R242E+I263L;

Y11V+D59N+I96L+S164G+L222I+G251D+L253V;

K19T+D59N+I96V+S164G+L166V+L222I+R242E+I263L;

Y11V+K19T+D59N+I96L+S164N+L166I+L222I+G251D;

K19T+I96L+S164G+L166V+L222I+R242E+N249D+G251D+I263V;

K19T+I96L+S164G+R242E+L253I;

K19S+D59N+E83S+I96L+S164N+L222I+G251D;

K19T+D59N+I96L+S164G+L222V+N249E+G251D+I263V;

K19T+D59N+I96L+S164G+L222I+N249E+G251D+L253V+I263L;

Y11I+K19T+I96L+S164G+L222V+R242E+G251D;

I96L+S164G+L222I+R242E+N249D+G251D+I263L;

K19T+D59N+I96L+S164G+L166I+L222I+R242D+G251D+I263V;

K19T+D59N+I96L+S164G+L222V+R242E+N249D+L253I;

H76Q+I96L+S164G+L222I+R242E+G251D+S273Y;

K19T+E83S+I96L+S164G+L222I+R242E+N249D+G251D+L253I;

I96L+S164G+L166V+L222I+R242E+N249D+I263L;

Y11V+K19T+E83S+I96L+S164G+L166V+L222I+R242E+G251D;

Y11V+K19T+I96L+S164G+L166V+L222I+R242E;

Y11V+E83S+I96L+S164G+L222I+R242E+G251D+L253I+I263L;

Y11V+I96L+S164G+L222I+R242E+N249D+L253I+I263L;

K19T+I96L+S164G+L166V+L222I+R242E+N249D+I263L;

Y11V+E83S+I96L+S164G+L222I+R242E+L253I+I263L;

K19T+E83S+I96L+S164G+L166V+L222I+R242E+N249D+G251D+L253I;

I96L+S164G+L222I+R242E+G251D+S274Y;

H76Q+I96L+S164G+L222I+R242E+G251D;

I96L+S164G+L222I+R242E+G251D; V32L+N100Q+N291Q;

V221K+N100Q+N291Q; D290E+N100Q+N291Q; V136I+N100Q+N291Q;

E240Q+N100Q+N291Q; R242Q+N100Q+N291Q; G289S+N100Q+N291Q;

N292H+N100Q+N291Q; L295K+N100Q+N291Q; V136E+N100Q+N291Q;

D290L+N100Q+N291Q; F119Y+N100Q+N291Q; Q280E+N100Q+N291Q;

F282E+N100Q+N291Q; R254S+N100Q+N291Q; R242E+N100Q+N291Q;

V203R+N100Q+N291Q; N249R+N100Q+N291Q; H56K+N100Q+N291Q;

S74D+N100Q+N219Q; A131D+N100Q+N291Q; Y190A+N100Q+N291Q;

I297A+N100Q+N291Q; H76Q+N100Q+N291Q; S273Y+N100Q+N291Q;

K19T+N100Q+N291Q; D59N+N100Q+N291Q; L222I+N100Q+N291Q;

V309I+N100Q+N291Q; I96L+N100Q+N291Q; Y21S+N100Q+N291Q;

L130I+N100Q+N291Q; S132A+N100Q+N291Q; S226T+N100Q+N291Q;

G251D+N100Q+N291Q; Y243E+N100Q+N291Q; S273D+N100Q+N291Q;

R242E+Q280E+N100Q+N291Q; R242E+N252D+N100Q+N291Q;

N252D+Q280E+N100Q+N291Q; Y243E+Q280E+N100Q+N291Q;

Y243E+N252D+N100Q+N291Q; R254E+Q280E+N100Q+N291Q;

S273D+Q280E+N100Q+N291Q; R242E+G251D+N100Q+N291Q;

R242E+G251D+Q280E+N100Q+N291Q;

R242E+S273D+Q280E+N100Q+N291Q;

N252D+S273D+Q280E+N100Q+N291Q;

G251D+S273D+Q280E+N100Q+N291Q;

R242E+R254E+Q280E+N100Q+N291Q;

R242E+R254E+S273D+Q280E+N100Q+N291Q;

Y243E+R254E+S273D+N100Q+N291Q; V223F+N252Q+N291Q;

E290D+N252Q+N291Q; A117S+N252Q+N291Q; I136V+N252Q+N291Q;

Q242R+N252Q+N291Q; Q278K+N252Q+N291Q; S289G+N252Q+N291Q;

Q294E+N252Q+N291Q; D249N+N252Q+N291Q; D251G+N252Q+N291Q;

G244D+N252Q+N291Q; Q56H+N252Q+N291Q; L32I+N252Q+N291Q;

K71E+N252Q+N291Q; P72T+N252Q+N291Q; Q83T+N252Q+N291Q;

V113F+N252Q+N291Q; E133S+N252Q+N291Q; Y134G+N252Q+N291Q;

K71A+N252Q+N291Q; Y11H+N100Q+N291Q; Y11K+N100Q+N291Q;

Y11R+N100Q+N291Q; Y11H+D290E+N100Q+N291Q;

Y11R+D290E+N100Q+N291Q; Y11F+N100Q+N291Q; Y11I+N100Q+N291Q;

Y11L+N100Q+N291Q; L12F+N100Q+N291Q; L12I+N100Q+N291Q;

D13N+N100Q+N291Q; D13Q+N100Q+N291Q; D13S+N100Q+N291Q;

D13T+N100Q+N291Q; D13F+N100Q+N291Q; D13L+N100Q+N291Q;

D13V+N100Q+N291Q; D13Y+N100Q+N291Q

R67Q+N100Q+L130I+M157L+V248I+N291Q;

N100Q+L130I+S132A+M157L+K231N;

R67Q+I96L+L130I+M157L+L222I+M256L;

R67Q+L130I+S132A+M157L+R242E+V248I;

R67Q+N100Q+M157L+R242E+M256L; R67Q+G70D+M157L+R242E+V248I;

V32L+R67Q+M157L+L222I+R242E; Y11V+R67Q+M157L+V248I+M256L;

R67Q+V136I+M157L+L222I+V248I; L130I+M157L+V248I+M256L+N291Q;

R67Q+I96L+L130I+M157L+K231N+R242E;

V32L+R67Q+L130I+M157L+L222I+K231N;

L130I+V136I+M157L+L222I+N292H; R67Q+G70D+M157L+L222I+N291Q;

V32L+R67Q+L130I+K231N+N292H;

Y11V+R67Q+N100Q+L130I+V136I+M157L;

R67Q+L130I+L222I+R242E+M256L; R67Q+M157L+L222I+V248I+N292H;

V32L+R67Q+M157L+M256L+N291Q;

R67Q+L130I+S132A+M157L+L222I+N292H;

R67Q+N100Q+L130I+M157L+K231N+N291Q;

R67Q+L130I+K231N+V248I+N291Q;

Y11V+R67Q+L130I+M157L+L222I+K231N;

I145V+L130I+M157L+K231N+R242E; V32L+R67Q+V136I+M157L+N291Q;

R67Q+N100Q+L130I+D158S+V248I;

I45V+R67Q+L130I+M157L+L222I+K231N;

V32L+R67Q+L130I+S132A+M157L+V248I;

Y11V+R67Q+L130I+M157L+N291Q+N292H;

R67Q+N100Q+L130I+M157L+L222I+K231N;

I45V+R67Q+G70D+L130I+S132A; I45V+R67Q+L130I+V248I+N292H;

Y11V+R67Q+L130I+M157L+L222I+R242E;

R67Q+N100Q+D158S+L130I+M157L+L222I;

R67Q+L130I+V136I+M157L+K231N+V248I;

I45V+R67Q+L130I+L222I+N291Q;

R67Q+G70D+L130I+M157L+K231N+M256L;

V32L+R67Q+L130I+M157L+D158S+V248I;

R67Q+L130I+M157L+D158S+R242E+N291Q;

R67Q+L130I+M157L+D158S+K231N+N292H;

R67Q+L130I+V248I+M256L+N292H; V32L+R67Q+I96L+L130I+M157L+V248I;

R67Q+I96L+N100Q+L130I+M157L+N292H;

V32L+R67Q+G70D+N100Q+M157L;

V32L+R67Q+L130I+M157L+K231N+M256L; R67Q+I96L+M157L+L222I+K231N;

R67Q+M157L+L222I+K231N+V248I;

R67Q+L130I+M157L+R242E+M256L+N292H; R67Q+L222I+K231N+V248I;

R67Q+S132A+L222I+K231N+R242E+V248I;

Y11V+K19T+D59N+S164G+L166V+L222I+R242E+N249E+G251D;

Y11V+K19T+D59N+I96L+S164G+L166I+L222I+R242E+N249E+G251D;

Y11I+K19T+D59N+I96L+S164G+L166V+L222I+R242E+N249E+G251D;

Y11I+K19T+D59N+I96L+S164G+L166I+L222I+R242E+G251D;

Y11V+K19T+D59N+I96L+L166V+L222V+R242E+N249E+G251D+L253I;

Y11V+K19T+D59N+I96L+S164G+L166V+R242E;

Y11V+K19T+D59N+I96L+S164G+L222V+R242E+G251D;

Y11V+K19T+D59N+I96L+S164G+L166I+R242E+N249E+G251D+L253I;

Y11V+K19T+D59N+I96L+S164G+L166V+L222V+R242E+N249E+G251D;

Y11V+K19T+D59N+I96L+S164G+L166I+L222V+R242E+N249E+G251D+L253I;

Y11V+K19T+D59N+L166V+L222I+R242E+N249E+G251D+L253I;

Y11V+K19T+D59N+I96L+S164G+L166V+L222I+R242E+N249E;

Y11V+K19T+D59N+S164G+L166I+L222I+R242E+G251D;

Y11V+K19T+D59N+I96L+S164G+R242E+G251D;

Y11V+D59N+I96L+S164G+L166I+L222V+R242E+G251D+L253I;

Y11V+D59N+I96L+S164G+L166I+L222I+R242E+G251D;

Y11I+D59N+I96L+S164G+L166V+L222I+R242E+G251D+L253I;

Y11V+K19T+D59N+I96L+S164G+L222I+R242E+N249E+G251D;

Y11V+K19T+D59N+I96L+S164G+L166I+L222V+R242E+G251D;

Y11V+K19T+D59N+I96L+S164G+L166V+L222V+R242E+N249E+L253I;

Y11V+K19T+D59N+I96L+S164G+L166I+L222V+R242E+N249E+G251D;

Y11I+K19T+I96L+S164G+L166V+R242E+N249E+G251D;

Y11V+K19T+D59N+I96L+S164G+L166V+L222V+R242E+G251D;

Y11V+K19T+D59N+I96L+S164G+L222V+R242E+N249E+G251D;

Y11I+K19T+L222V+R242E+N249E+G251D;

Y11V+K19T+I96L+L222V+R242E+N249E+G251D;

Y11I+K19T+D59N+I96L+S164G+L166V+L222V+R242E+N249E+G251D;

Y11V+K19T+I96L+S164G+L166V+L222V+R242E+N249E+G251D;

Y11I+K19T+D59N+I96L+S164G+L166I+L222V+R242E+N249E+G251D;

Y11I+I96L+S164G+L166V+L222V+R242E+N249E+G251D;

Y11I+K19T+D59N+I96L+S164G+L222V+R242E+N249E;

Y11I+K19T+D59N+I96L+L222V+R242E+N249E+G251D;

Y11I+K19T+D59N+I96L+S164G+L222I+R242E;

Y11I+K19T+D59N+I96L+S164G+L166V+R242E+G251D;

Y11I+K19T+D59N+S164G+L166I+L222V+R242E+G251D;

Y11I+I96L+L222V+R242E+N249E+G251D; Y11I+I96L+S164G+L222I+R242E;

Y11V+K19T+I96L+L166V+L222V+R242E+G251D;

Y11I+D59N+I96L+S164G+L222I+R242E+G251D;

Y11I+D59N+I96L+S164G+L222V+R242E+N249E+G251D;

Y11I+K19T+D59N+I96L+S164G+L222I+R242E+N249E+G251D;

Y11I+D59N+I96L+S164G+L166V+L222V+R242E+G251D;

Y11V+K19T+D59N+I96L+L222V+R242E+G251D;

Y11I+K19T+5164G+L166I+L222V+R242E+N249E+G251D or

Y11I+D59N+I96L+S164G+L166V+L222V+R242E+N249E+G251D.

An alternative aspect relates to methods for making a food or feed product comprising adding an effective amount of the isolated chymosin polypeptide variant of present invention to the food or feed ingredient(s) and carrying our further manufacturing steps to obtain the food or feed product, such as e.g. a milk-based product and optionally more specifically methods for making cheese such as e.g. Pasta filata, Cheddar, Continental type cheeses, soft Cheese or White Brine Cheese.

Accordingly, present invention relates to a food or feed product comprising a chymosin polypetide variant as described herein.

The polypeptide variant of present invention may also be used to reduce bitterness in cheese and other dairy products as e.g. yoghurt.

In cheese ripening, chymosin cleaves β-casein primarily between Leu192 and Tyr193 (references 2,3). The resulting peptide β(193-209) will be further degraded by proteases to short hydrophobic peptides that taste bitter (reference 4). Since bitterness in dairy applications is most often considered an undesirable feature, it is desirable to develop chymosin variants with lower β-casein cleavage frequency.

Based on intelligent design and a comparative analysis of different variants the present inventors identified a number of amino acid positions that are herein important in the sense that by making a variant in one or more of these positions one may get an improved chymosin variant with a lower β-casein cleavage frequency.

The amino acid numbering as used herein to specify a variant or mutation is done on the mature peptide numbering. For clarification, the mature polypeptide of SEQ ID NO:2 corresponds to SEQ ID NO:4.

As known in the art—different natural wildtype chymosin polypeptide sequences obtained from different mammalian species (such as e.g. bovines, camels, sheep, pigs, or rats) are having a relatively high sequence similarity/identity.

In FIG. 1 this is exemplified by an alignment of herein relevant different chymosin sequences.

In view of this relatively close sequence relationship—it is believed that the 3D structures of different natural wildtype chymosins are also relatively similar.

In the present context—a natural obtained wildtype chymosin (such as bovine chymosin or camel chymosin) may herein be an example of a parent polypeptide—i.e. a parent polypeptide to which an alteration is made to produce a variant chymosin polypeptide of the present invention.

Without being limited to theory—it is believed that the herein discussed chymosin related amino acid positions are of general importance in any herein relevant chymosin enzyme of interest (e.g. chymosins of e.g. bovines, camels, sheep, pigs, rats etc)—in the sense that by making a variant in one or more of these positions one may get an improved chymosin variant in general (e.g. an improved bovine, camel, sheep, pig or rat chymosin variant).

As discussed herein—as a reference sequence for determining the amino acid position of a parent chymosin polypeptide of interest (e.g. camel, sheep, bovine etc) is herein used the public known mature Camelius dromedarius chymosin sequence of SEQ ID NO:2 herein. It may herein alternatively be termed camel chymosin. The sequence is also shown in FIG. 1 herein.

In the present context it is believed that a parent chymosin polypeptide (e.g. from sheep or rat) that has at least 65% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin) may herein be seen as sufficient structural related to e.g. bovine or camel chymosin in order to be improved by making a variant in any of the amino acid positions as described herein.

Embodiments of the present invention are described below.

DEFINITIONS

All definitions of herein relevant terms are in accordance of what would be understood by the skilled person in relation to the herein relevant technical context.

The term “β-cleavage” or “cleavage of β-casein” means any enzymatic cleavage of β-casein. Such as e.g. cleavage between Leu192 and Tyr193, resulting in the formation of β(193-209) peptide. In one aspect β-cleavage is determined by quantifying the β(193-209) peptide obtained by incubating skim milk with the chymosin variant polypeptide or the camel chymosin, wherein quantification is carried out by RP-HPLC coupled to an ESI-Q-TOF mass spectrometer. Full details of a preferred method of determining β-casein cleavage are described in the Examples.

The term “chymosin” relates to an enzyme of the EC 3.4.23.4 class. Chymosin has a high specificity and predominantly clots milk by cleavage of a single 104-Ser-Phe-|-Met-Ala-108 bond in κ-chain of casein. As a side-activity, chymosin also cleaves β-casein primarily between Leu192 and Tyr193 (references 2,3). The resulting peptide β(193-209) will be further degraded by proteases to short hydrophobic peptides that taste bitter (reference 4). An alternative name of chymosin used in the art is rennin.

The term “chymosin activity” relates to chymosin activity of a chymosin enzyme as understood by the skilled person in the present context.

The skilled person knows how to determine herein relevant chymosin activity.

The term “specific clotting activity” describes the milk clotting activity of a chymosin polypeptide and can be determined according to assays well known in the art. A preferred method for determining the specific clotting activity in terms of IMCU/mg of protein is the standard method developed by the International Dairy Federation (IDF method), which comprises steps, wherein milk clotting activity is determined from the time needed for a visible flocculation of a milk substrate and the clotting time of a sample is compared to that of a reference standard having known milk-clotting activity and the same enzyme composition by IDF Standard 110B as the sample. Samples and reference standards are measured under identical chemical and physical conditions. Full details of a the IDF method are described in the Examples.

As known in the art—the herein relevant so-called C/P ratio is determined by dividing the specific clotting activity (C) with the proteolytic activity (P).

As known in the art—a higher C/P ratio implies generally that the loss of protein during e.g. cheese manufacturing due to non-specific protein degradation is reduced, i.e. the yield of cheese is improved.

The term “isolated variant” means a variant that is modified by the act of man. In one aspect, the variant is at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS PAGE.

The amino acid numbering as used herein to specify chymosin polypeptide variants of the present invention is done on the mature peptide numbering. In the sequence listing provided with the present application:

SEQ ID NO:1 represents the complete polypeptide sequence of bovine preprochmyosin;

SEQ ID NO:2 represents the complete polypeptide sequence of camel preprochmyosin;

SEQ ID NO:3 represents the polypeptide sequence of mature bovine chymosin;

SEQ ID NO:4 represents the polypeptide sequence of mature camel chymosin.

In other words, SEQ ID NOs:3 and 4 correspond to amino acids 59 to 381 of SEQ ID NOs:1 and 2, respectively. All of the specific substitutions identified herein are identified in relation to the position of the mature chymosin sequence, i.e. in relation to the amino acid numbering of SEQ ID NOs:3 or 4. Insofar as the position is identified in relation to the amino acid numbering of SEQ ID NOs:1 or 2 one has to subtract 58 residues to identify the position in SEQ ID NOs:3 or 4 and vice versa.

The term “mature polypeptide” means a peptide in its final form following translation and any post-translational modifications, such as N terminal processing, C terminal truncation, glycosylation, phosphorylation, etc. In the present context may a herein relevant mature chymosin polypeptide be seen as the active chymosin polypeptide sequence—i.e. without the pre-part and/or pro-part sequences. Herein relevant examples of a mature polypeptide are e.g. the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO:2 (camel chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO:2.

The term “parent”, “parent polypeptide” or “parent polypeptide having chymosin activity” means a polypeptide to which an alteration is made to produce the enzyme variants of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant thereof. In a preferred embodiment of present invention, the parent polypeptide has at least 80%, such as at least e.g. 85%, 95%, 97%, 98%, 99% or 100% sequence identity with the polypeptide of SEQ ID NO:4 (camel chymosin).

The term “Sequence Identity” relates to the relatedness between two amino acid sequences or between two nucleotide sequences.

For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment).

The term “variant” means a peptide having chymosin activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position.

The amino acid may be natural or unnatural amino acids—for instance, substitution with e.g. a particularly D-isomers (or D-forms) of e.g. D-alanine could theoretically be possible.

The term “wild-type” peptide refers to a nucleotide sequence or peptide sequence as it occurs in nature, i.e. nucleotide sequence or peptide sequence which hasn't been subject to targeted mutations by the act of man.

DRAWINGS

FIG. 1: An alignment of herein relevant different chymosin sequences. The shown “Bos_bovis_chymosin B” is bovine chymosin of SEQ ID NO: 1 herein and the shown “Camelus_dromedarius” is camel chymosin of SEQ ID NO:2 herein. Using bovine chymosin of SEQ ID NO: 1 as reference sequence as described herein is can e.g. be seen that bovine chymosin has “V” in position 10 and camel chymosin has “A” in the same position 10. It may e.g. also be seen that bovine/Rat have “Q” in position 352 and Camel/C._bactrianus have “E” in the same position 352.

In relation to the chymosin sequences shown in FIG. 1—sheep has 94.5% sequence identity with bovine SEQ ID NO: 1; C._bactrianus has 83.2% sequence identity with bovine SEQ ID NO: 1; Camelus_dromedarius (camel chymosin of SEQ ID NO:2) has 84% sequence identity with bovine SEQ ID NO: 1; pig has 80.3% sequence identity with bovine SEQ ID NO: 1 and rat has 71.9% sequence with bovine identity SEQ ID NO: 1.

As understood by the skilled person in the present context—herein relevant sequence identity percentages of mature polypeptide sequences of e.g. sheep, C._bactrianus, camel, pig or rat chymosin with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin—i.e. amino acid positions 59 to 381 of SEQ ID NO: 1) are relatively similar to above mentioned sequence identity percentages.

FIGS. 2 and 3:

3D structure of camel chymosin (PDB: 4AA9) with a model of bound β-casein shown in purple. The β-casein is placed in the chymosin substrate binding cleft with the scissile bond between residues 192 and 193. Camel chymosin residues V32, L130, and S132 are highlighted in green.

FIG. 4:

3D structure of camel chymosin (PDB: 4AA9). Camel chymosin residues V32 and L12 are highlighted in green.

DETAILED DESCRIPTION OF THE INVENTION

Determining the Amino Acid Position of a Chymosin of Interest

As discussed above—as a reference sequence for determining the amino acid position of a herein relevant chymosin polypeptide of interest (e.g. camel, sheep, bovine etc.) is herein used the public known camel chymosin sequence disclosed as SEQ ID NO:2 herein.

The amino acid sequence of another chymosin polypeptide is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the ClustalW algorithm as described in working Example 1 herein.

Based on above well-known computer programs—it is routine work for the skilled person to determine the amino acid position of a herein relevant chymosin polypeptide of interest (e.g. camel, sheep, bovine etc.).

In FIG. 1 herein is shown an example of an alignment.

Just as an example—in FIG. 1 can e.g. be seen that herein used bovine reference SEQ ID NO: 1 has a “G” in position 50 and “Camelus_dromedarius” (SEQ ID NO:2 herein) has an “A” in this position 50.

Nomenclature of Variants

In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IUPAC single letter or three letter amino acid abbreviations are employed.

The specific variants discussed in this “nomenclature” section below may not be herein relevant variants of the present invention—i.e. this “nomenclature” section is just to describe the herein relevant used nomenclature as such. As indicated above, the amino acid numbering used to specify chymosin polypetide variants of the present invention is based on the position of the amino acid in the mature chymosin polypeptide sequence.

Substitutions. For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid.

Accordingly, a theoretical substitution of threonine with alanine at position 226 is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively. A substitution e.g. designated “226A” refers to a substitution of a parent amino acid (e.g. T, Q, S or another parent amino acid) with alanine at position 226. Likewise, a substitution designated “A226” or “A226X” refers to a substitution of an alanine in position 226 with another unspecified amino acid.

Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple alterations. Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of tyrosine and glutamic acid for arginine and glycine at positions 170 and 195, respectively.

Different substitutions. Where different substitutions can be introduced at a position, the different substitutions are separated by a comma, e.g., “Arg170Tyr,Glu” or “R170Y,E” represents a substitution of arginine with tyrosine or glutamic acid at position 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” or “Y167G,A+R170G,A” designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Preferred Variants:

As outlined in the Examples below, the inventors have made a number of preferred chymosin polypeptide variants that cleave β-casein with a lower frequency than the corresponding parent polypeptide while at least maintaining its clotting activity.

Preferred Variants with Reduced β-Casein Cleavage Frequency:

The isolated chymosin polypeptide variants of the present invention have a specific clotting activity (IMCU/mg total protein) that is at least 80% of the specific clotting activity of isolated camel chymosin polypeptide characterized by SEQ ID NO:4, including a specific clotting activity (IMCU/mg total protein) that is at least 85%, at least 90%, at least 95% or at least 97% of the specific clotting activity of isolated camel chymosin polypeptide characterized by SEQ ID NO:4.

The isolated chymosin polypeptide variant of present invention may be derived from a parent polypeptide has at least 80%, such as at least e.g. 80%, 85%, 95%, 97%, 98%, 99% sequence identity with the polypeptide of SEQ ID NO:4 (camel chymosin).

The isolated chymosin polypeptide variant of present invention may comprise one or more amino acid substitutions, deletions or insertions, wherein the one or more substitution, deletion or insertion is specified in relation to the amino acid sequence of SEQ ID NO:4: Y11, L130, S132, V32, S226, R266, L12, V221, S255, S277, L222, L253, M157, V260, S271, H76, K19, V183, S164, I263, V51, T239, Y307, R67, G251, R61, Q288, E83, D59, V309, S273, G251, S154, Y21, V203, L180, E294, G289, L215, D144, I303, L105, T284, Y127, V248, K321, V205, E262, K231, R316, M256, D158, D59, N249, L166, R242 or I96 such as e.g. Y11I, Y11V, L130I, S132A, V32L, S226T, R266V, L12M, V221M, S255Y, S277N, L222I, L253I, M157L, V260T, S271P, H76Q, K19T, V183I, S164G, I263L, V51L, T239S, Y307F, R67Q, G251D, R61Q, Q288E, E83S, D59N, V309I, S273Y, G251W, S154A, Y21S, V203A, L180I, E294Q, G289S, L215V, D144Q, I303L, L105E, T284S, Y127F, V248I, K321P, V205I, E262T, K231N, R316L, M256L, D158S, D59N, N249E, L166V, R242E and/or I96L.

In a related aspect, the isolated chymosin polypeptide variant of present invention may comprise a combination of substitutions, wherein the combination of substitutions is selected from a list comprising:

Y11+K19+D59+I96+S164+L166+L222+R242+N249+G251; Y11+K19+D59+I96+S164+L222+R242+G251; Y11+K19+D59+I96+S164+L166+R242+N249+G251+L253; Y11+K19+I96+S164+L166+R242+N249+G251; Y11+K19+D59+I96+S164+L222+R242+N249+G251; Y11+K19+I96+S164+L166+L222+R242+N249+G251; Y11+K19+D59+I96+S164+L222+R242+N249; Y11+K19+D59+I96+S164+L166+R242+G251; Y11+I96+S164+L222+R242; Y11+D59+I96+S164+L222+R242+G251 or Y11I+K19+D59+I96+S164+ +R242+N249+G251 such as e.g. Y11I+K19T+D59N+I96L+S164G+L166V+L222I+R242E+N249E+G251D; Y11V+K19T+D59N+I96L+S164G+L222V+R242E+G251D; Y11V+K19T+D59N+I96L+S164G+L166I+R242E+N249E+G251D+L253I; Y11I+K19T+I96L+S164G+L166V+R242E+N249E+G251D; Y11V+K19T+D59N+I96L+S164G+L222V+R242E+N249E+G251D; Y11V+K19T+I96L+S164G+L166V+L222V+R242E+N249E+G251D; Y11I+K19T+D59N+I96L+S164G+L222V+R242E+N249E; Y11I+K19T+D59N+I96L+S164G+L166V+R242E+G251D; Y11I+I96L+S164G+L222I+R242E; Y11I+D59N+I96L+S164G+L222I+R242E+G251D or Y11I+K19T+D59N+I96L+S164G+L222I+R242E+N249E+G251D and wherein each substitution is specified in relation to the amino acid sequence of SEQ ID NO:4.

In a related aspect, the variant may comprise alterations in one or more specified positions compared to a parent polypeptide having chymosin activity, wherein in the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions 11, 130, 132, 32, 226, 266, 12, 221, 255, 277, 222, 253, 157, 260, 271, 76, 19, 183, 164, 263, 51, 239, 307, 67, 251, 61, 288, 83, 59, 309, 273, 251, 154, 21, 203, 180, 294, 289, 215, 144, 303, 105, 284, 127, 248, 321, 205, 262, 231, 316, 256, 158, 59, 249, 166, 242 or 96, wherein the amino acid position of the parent polypeptide is determined by an alignment of the parent polypeptide with the mature polypeptide of SEQ ID NO:2 (camel chymosin) and the parent polypeptide has at least 65% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin), which is from amino acid position 59 to amino acid position 381 of SEQ ID NO:2, wherein the isolated chymosin polypeptide variant cleaves β-casein with a lower frequency than the corresponding parent polypeptide.

In a preferred embodiment the parent polypeptide has at least 80%, such as at least e.g. 85%, 95%, 97%, 98%, 99% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin).

Preferably, an isolated chymosin polypeptide variant as described herein is a variant, wherein the variant has a lower β-casein cleavage frequency as compared to the parent peptide from which the variant is derived.

More preferably, an isolated chymosin polypeptide variant as described herein is a variant, wherein the variant has

-   -   a chymosin activity giving lower β-casein cleavage frequency as         compared to the bovine chymosin comprising the mature         polypeptide of SEQ ID NO: 1 herein; and     -   a chymosin activity giving a lower β-casein cleavage frequency         as compared to the camel chymosin comprising the mature         polypeptide of SEQ ID NO:2 herein.

As discussed above—as a reference sequence for determining the amino acid position of a herein relevant chymosin polypeptide of interest (e.g. camel, sheep, bovine etc.) is herein used the mature peptide of the publicly known camel chymosin sequence disclosed as SEQ ID NO:2 herein.

As discussed above—based on e.g. the computer sequence alignment programs discussed herein—it is routine work for the skilled person to determine the here—in relevant amino acid position of a herein relevant chymosin polypeptide of interest (e.g. camel, sheep, bovine etc.).

The term “the parent polypeptide has at least 65% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin)” may be seen as relating to a sequence based limitation of the parent chymosin polypeptide used to make a herein relevant variant thereof.

In a preferred embodiment—the parent polypeptide has at least 92% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin), more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin) and even more preferably the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin). It may be preferred that the parent polypeptide is the mature polypeptide of SEQ ID NO:2 (Camel chymosin).

As understood by the skilled person in the present context—a herein relevant parent polypeptide having chymosin activity may already e.g. be a variant of e.g. a corresponding wildtype chymosin.

Said in other words, a herein relevant isolated chymosin polypeptide variant may comprise alterations (e.g. substitutions) in other positions than the positions claimed herein.

In relation to the chymosin sequences shown in FIG. 1 herein—sheep has 94.5% sequence identity with bovine SEQ ID NO: 1; C._bactrianus (camel) has 83.2% sequence identity with bovine SEQ ID NO: 1; pig has 80.3% sequence identity with bovine SEQ ID NO: 1 and rat has 71.9% sequence with bovine identity SEQ ID NO: 1.

As understood by the skilled person in the present context—herein relevant sequence identity percentages of e.g. mature sheep, C._bactrianus, camel, pig or rat chymosin with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin—i.e. amino acid positions 59 to 381 of SEQ ID NO: 1) are relatively similar to above mentioned sequence identity percentages.

Preferably, an isolated bovine chymosin polypeptide variant as described herein is a variant, wherein the variant has a chymosin activity giving a lower β-casein cleavage frequency as compared to the β-casein cleavage frequency of camel chymosin comprising the mature polypeptide of SEQ ID NO:2.

As discussed above—in working examples herein were made variants using the polypeptide of SEQ ID NO:2 (camel chymosin) as parent polypeptide—such variant may herein be termed camel chymosin variant.

As understood by the skilled person in the present context—an isolated chymosin variant may comprise alterations (e.g. substitutions) in other amino acid positions than given above.

For instance, a camel chymosin variant with e.g. 5-10 alterations (e.g. substitutions) as compared to wildtype camel chymosin polypeptide of SEQ ID NO:2 will still be a parent polypeptide that has at least 95% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin).

It may be preferred that the isolated camel chymosin variant comprises less than 30 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO:2 (camel chymosin) or it may be preferred that the isolated camel chymosin variant comprises less than 20 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO:2 (camel chymosin) or it may be preferred that the isolated camel chymosin variant comprises less than 10 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO:2 (camel chymosin) or it may be preferred that the isolated camel chymosin variant comprises less than 5 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO:2 (camel chymosin).

As understood by the skilled person in the present context—the term “the isolated variant polypeptide has less than 100% sequence identity with the mature polypeptide of SEQ ID NO:2 (camel chymosin)” above relates to that the herein described isolated camel chymosin variant shall not have a polypeptide sequence that is 100% identical to the public known wildtype camel chymosin sequence of SEQ ID NO:2.

A preferred embodiment relates to an isolated camel chymosin polypeptide variant, wherein the alteration comprises a substitution, a deletion or an insertion in at least one amino acid position corresponding to any of positions claimed herein.

It may be preferred that at least one alteration is a substitution—i.e. a herein relevant preferred embodiment relates to an isolated chymosin polypeptide variant, wherein the alteration is comprising a substitution in at least one amino acid position corresponding to any of positions claimed herein.

Preferred parent polypeptide having chymosin activity:

Preferably, the parent polypeptide has at least 80%, such as e.g. 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) and/or SEQ ID NO:2 (camel chymosin).

Just as an example—a herein suitable relevant parent polypeptide could e.g. be bovine chymosin A—as known in the art bovine chymosin A may only have one amino acid difference as compared to bovine chymosin B of SEQ ID NO: 1 herein.

In a preferred embodiment—the parent polypeptide has at least 90% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) and even more preferably the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin). It may be preferred that the parent polypeptide is the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

As understood by the skilled person in the present context—a herein relevant parent polypeptide having chymosin activity may already e.g. be a variant of e.g. a corresponding wildtype chymosin.

For instance, a bovine chymosin variant with e.g. 5-10 alterations (e.g. substitutions) as compared to mature wildtype bovine chymosin polypeptide of SEQ ID NO: 1 will still be a parent polypeptide that has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovine chymosin).

Said in other words and in general—a herein relevant isolated chymosin polypeptide variant may comprise alterations (e.g. substitutions) in other positions than the positions claimed herein.

As understood by the skilled person in the present context—a parent polypeptide that has at least 90% sequence identity with the mature polypeptide of SEQ ID NO:2 (Camel) is still within the SEQ ID NO: 1 (Bovine) based sequence identity requirement i.e. it will be a parent polypeptide that has at least 65% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

In a preferred embodiment—the parent polypeptide has at least 92% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin), more preferably the parent polypeptide has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) and even more preferably the parent polypeptide has at least 97% sequence identity with the mature polypeptide of SEQ ID NO: 1 (bovine chymosin). It may be preferred that the parent polypeptide is the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

As understood by the skilled person in the present context—an isolated chymosin variant may comprise alterations (e.g. substitutions) in other amino acid positions than given above.

For instance, a bovine chymosin variant with e.g. 5-10 alterations (e.g. substitutions) as compared to wildtype bovine chymosin polypeptide of SEQ ID NO: 1 will still be a parent polypeptide that has at least 95% sequence identity with the mature polypeptide of SEQ ID NO: 1 (Bovine chymosin).

It may be preferred that the isolated bovine chymosin variant comprises less than 30 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) or it may be preferred that the isolated bovine chymosin variant comprises less than 20 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) or it may be preferred that the isolated bovine chymosin variant comprises less than 10 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin) or it may be preferred that the isolated bovine chymosin variant comprises less than 5 amino acid alterations (e.g. substitutions) as compared to the mature polypeptide of SEQ ID NO: 1 (bovine chymosin).

The camel chymosin polypeptide of SEQ ID NO:2 has 84% sequence identity with the bovine polypeptide of SEQ ID NO: 1 (i.e. the complete SEQ ID NO: 1 from position 1 to 381, which includes pre and pro sequence).

A Method for Making an Isolated Chymosin Polypeptide Variant

As discussed above—as known in the art, the skilled person may, based on his common general knowledge, routinely produce and purify chymosin and chymosin variants.

Said in other words, once the skilled person is in possession of a herein relevant parent polypeptide having chymosin activity of interest (e.g. from bovines, camels, sheep, pigs, or rats) it is routine work for the skilled person to make a variant of such a parent chymosin of interest.

An example of a suitable method to produce and isolate a chymosin (variant or parent) may be by well-known e.g. fungal recombinant expression/production based technology as e.g. described in WO02/36752A2 (Chr. Hansen).

It is also routine work for the skilled person to make alteration at one or more positions in a parent polypeptide having chymosin activity, wherein the alteration is comprising a substitution, a deletion or an insertion in at least one amino acid position.

As known to the skilled person this may e.g. be done by so-called site directed mutagenesis and recombinant expression/production based technology.

It is also routine work for the skilled person to determine if a herein relevant parent polypeptide (e.g. camel or bovine wildtype chymosin) and/or a herein relevant variant has chymosin activity or not. As known in the art chymosin specificity may be determined by the so-called C/P ratio, which is determined by dividing the specific clotting activity (C) with the proteolytic activity (P).

As known in the art—a higher C/P ratio implies generally that the loss of protein during e.g. cheese manufacturing due to non-specific protein degradation is reduced, i.e. the yield of cheese is improved.

As also known in the art, β-casein cleavage and β-casein (including β(193-209)) formation may be determined using standard methods available to the person skilled in the art.

A Method for Making a Milk Based Product

As discussed above—an isolated chymosin polypeptide variant as described herein may be used according to the art—e.g. to make a milk based product of interest (such as e.g. a cheese product).

As discussed above—an aspect of the invention relates to a method for making a food or feed product comprising adding an effective amount of the isolated chymosin polypeptide variant as described herein to the food or feed ingredient(s) and carrying our further manufacturing steps to obtain the food or feed product.

Preferably, the food or feed product is a milk based product and wherein the method comprises adding an effective amount of the isolated chymosin polypeptide variant as described herein to milk and carrying our further manufacturing steps to obtain the milk based product.

For example, the chymosin polypeptide variant of the present invention may be added to a milk-based product after fermentation of the milk. In one aspect the chymosin polypeptide variant of the present invention is added for coagulation of a fermented milk product as part of a method of producing cheese.

The milk may e.g. be soy milk, sheep milk, goat milk, buffalo milk, yak milk, lama milk, camel milk or cow milk.

The milk based product may e.g. be a fermented milk product such as a quark or a cheese.

Food and Feed Products

The present invention also provides food and feed products comprising a chymosin polypetide variant of the present invention or a chymosin polypeptide variant obtainable according to a method of the present invention. The food and feed product is preferably a fermented food product, such as a fermented milk product, including cheese and quark.

In yet a related aspect, the present invention relates to a method for making a food or feed product comprising adding an effective amount of the isolated chymosin polypeptide variant according to the invention. Preferably, the food or feed product is a milk-based product.

The chymosin polypetide variant of present invention may also be used in a process for making cheese, such as e.g. to reduce bitterness in cheese.

EXAMPLES Example 1 Alignment and Numbering of Chymosin Protein Sequences and Variant Sequences

Chymosin protein sequences were aligned using the ClustalW algorithm as provided by the EBI (EBI, tools, multiple sequence alignment, CLUSTALW″, http://www.ebi.ac.uk/Tools/msa/clustalw2/) and as described in Larkin M A, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H, Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D, Gibson T J, Higgins D G (2007). Bio-informatics 23(21), 2947-2948.

ClustalW2 settings for multiple sequence alignments were Protein weight Matrix=BLOSUM, GAP open=10, GAP EXTENSION=0.05, GAP DISTANCES=8, No End Gaps, ITERATION=none, NUMITER=1, CLUSTERING=NJ

As a reference sequence the bovine chymosin B preprochymosin was used (Gen-bank accession number P00794—disclosed herein as SEQ ID NO: 1), where the N-terminal Methionin has number 1 (MRCL) and the C-terminal Isoleucin (in the protein sequence . . . LAKAI) has number 381.

Example 2 Design of Chymosin Variants

Chymosin variants were designed using different strategies.

When there is referred to camel chymosin there is referred to camel chymosin comprising the mature polypeptide of SEQ ID NO:2 herein.

Camel chymosin of SEQ ID NO:2 may be seen as a herein relevant parent polypeptide having chymosin activity used to make camel chymosin variants thereof. When there is referred to bovine chymosin there is referred to bovine chymosin comprising the polypeptide of SEQ ID NO: 1 herein.

Bovine chymosin of SEQ ID NO: 1 may be seen as a relevant parent polypeptide having chymosin activity used to make bovine chymosin variants thereof.

Variants 1 to 269 and 367 to 461 of camel chymosin were designed based on an alignment of a large set of public known aspartic protease sequences having an identity of 25% or more compared to bovine chymosin B.

Variations were generally introduced in regions with a high level of amino acid variation between species, while conserved regions were not changed. Amino acid substitutions were chosen based on phylogenetic, structural and experimental information to identify changes with high probability to show beneficial effects on β-casein cleavage. Multiple variations were introduced in each variant construct, ensuring that each single mutation was present in multiple variant constructs to minimize the effect of covariation between various substitutions. Machine learning and statistical analysis of experimental data were used to determine the relative contributions of the amino acid substitutions to measured coagulant performance of the chymosin variants (references 14, 15).

Variants 271 to 366 were designed based on detailed structural analysis of bovine chymosin (PDB code: 4AA8) and camel chymosin (PDB code: 4AA9). Variations were chosen based on the chemical nature of the respective amino acid side chains and their expected impact on either casein substrate binding or general enzyme properties. Most of the amino acid substitutions in variants 271 to 346 were made in sequence positions either within or in close structural proximity to the substrate binding cleft, or in secondary structural elements that get into contact with the bound casein substrate. Furthermore, changes were made in positions on the protein surface that alter the charge profile of these regions (reference 5) and are therefore expected to have an impact on enzyme performance. Variants 347 to 366 were made based on the different structural conformation of the N-terminal sequence in bovine and camel chymosin. Amino acid substitutions were made in positions within the substrate binding cleft that interact with the N-terminus in camel chymosin.

Example 3 Preparation of Chymosin Variant Enzyme Material

All chymosin variants were synthesized as synthetic genes and cloned into a fungal expression vector such as e.g. pGAMpR-C (described in WO02/36752A2)

The vectors were transformed into E. coli and plasmid DNA was purified using standard molecular biology protocols, known to the person skilled in the art.

The variant plasmids were individually transformed into an Aspergillus niger or Aspergillus nidulans strain and protein was produced essentially as described in WO02/36752A2 and purified using standard chromatography techniques.

As known in the art—the skilled person may, based on his common general knowledge, produce and purify chymosin and chymosin variants—such as herein described bovine and camel chymosin variants.

Example 4 Determination of Specific Chymosin Activity

4.1 Determination of Milk Clotting Activity

Milk clotting activity was determined using the REMCAT method, which is the standard method developed by the International Dairy Federation (IDF method) Milk clotting activity is determined from the time needed for a visible flocculation of a standard milk substrate prepared from a low-heat, low fat milk powder with a calcium chloride solution of 0.5 g per liter (pH 6.5). The clotting time of a rennet sample is compared to that of a reference standard having known milk-clotting activity and having the same enzyme composition by IDF Standard 110B as the sample. Samples and reference standards were measured under identical chemical and physical conditions. Variant samples were adjusted to approximately 3 IMCU/ml using an 84 mM acetic acid buffer pH 5.5. Hereafter, 200 μl enzyme preparation was added to 10 ml preheated milk (32° C.) in a glass test tube placed in a water bath, capable of maintaining a constant temperature of 32° C.±1° C. under constant stirring. Alternatively, 20 μL enzyme preparation was added to 1 mL preheated milk as described above.

The total milk-clotting activity (strength) of a rennet was calculated in International Milk-Clotting Units (IMCU) per ml relative to a standard having the same enzyme composition as the sample according to the formula:

${{Strength}\mspace{14mu}{in}\mspace{14mu}{IMCU}\text{/}{ml}} = \frac{{Sstandard} \times {Tstandard} \times {Dsample}}{{Dstandard} \times {Tsample}}$

Sstandard: The milk-clotting activity of the international reference standard for rennet.

Tstandard: Clotting time in seconds obtained for the standard dilution.

Dsample: Dilution factor for the sample

Dstandard: Dilution factor for the standard

Tsample: Clotting time in seconds obtained for the diluted rennet sample from addition of enzyme to time of flocculation

For clotting activity determination of library 1, 3, 4 and 6 variants as well as variants by structural design, the μIMCU method was used instead of the REMCAT method. As compared to REMCAT, flocculation time of chymosin variants in the μIMCU assay was determined by OD measurements in 96-well microtiter plates at 800 nm in a UV/VIS plate reader. A standard curve of various dilutions of a reference standard with known clotting strength was recorded on each plate. Samples were prepared by diluting enzyme in 84 mM acetate buffer, 0.1% triton X-100, pH 5.5. Reaction at 32° C. was started by adding 250 uL of a standard milk substrate containing 4% (w/w) low-heat, low fat milk powder and 7.5% (w/w) calcium chloride (pH≈6.5) to 25 uL enzyme sample. Milk clotting activity of chymosin variants in International Milk-Clotting Units (IMCU) per ml was determined based on sample flocculation time relative to the standard curve.

4.2 Determination of Total Protein Content

Total protein content was determined using the Pierce BCA Protein Assay Kit from Thermo Scientific following the instructions of the providers.

4.3 Calculation of Specific Clotting Activity

Specific clotting activity (IMCU/mg total protein) was determined by dividing the clotting activity (IMCU/ml) by the total protein content (mg total protein per ml).

Example 5 Determination of β-Casein Cleavage

Determination of β-Casein Hydrolysis Activity

Chymosin mediated proteolysis of milk proteins was characterized by determining profiles of water soluble peptides extracted at pH 4.6. A culture free cheese model made in 96 well plates was used for the study. In brief, 750 μl skim milk from Øllingegård, Denmark added glucono-delta-lactone (GDL) and calcium chloride was aliquoted into the wells of a 96 deep well plate. After 10 min from addition of GDL to the milk, variants of chymosin were added to individual wells of the plate to a final activity of 0.05 IMCU/ml. The formed coagulum was cut after 30 min from addition of rennet by thoroughly stirring the coagulum with a pipette tip; a new tip was used for each well. Subsequently, the plate was left for another 60 min before curd and whey was separated by centrifugation of the plate for 10 min at 2500 g. The milk was kept at 30° C. during renneting, cutting and syneresis. Finally, whey was decanted from the plate and the pellet of rennet curd left in the plate was stored for 4 days at room temperature. Peptides were extracted by adding 500 μl of 0.5 M tri-sodium citrate to each well and gentle shaking the plate for 24 hours at 37° C. The now fully dissolved rennet curd was then precipitated by adding hydrochloric acid to a final pH of 4.4-4.5. The plate was spun down in a centrifuge and the supernatant recovered for further analysis of pH 4.5 soluble peptides.

Profiles of pH 4.5 soluble peptides were determined using RP-HPLC coupled to an ESI-Q-TOF mass spectrometer. The analysis was performed by using a liquid chromatography system (Agilent 1290 infinity, Agilent Technologies A/S, Santa Clara, Calif., USA) coupled to a mass spectrometer (G6540A Q-TOF, Agilent Technologies A/S, Santa Clara, Calif., USA). The column in the LC system was Ascentis Express Peptide ES-C18m, 2.7 μm, 100×2.1 mm (Supelco, Sigma-Aldrich, St. Louis, USA). The mobile phase consisted of eluent A (0.1% formic acid in water) and eluent B (Acetonitrile: 0.1% formic acid in water, 9:1). After equilibration of the column with 2% B, a sample volume of 10 μL was injected. The peptides were separated by gradient elution generated by increasing eluent B from 2% to 50% over 15 column volumes. The flow rate was 0.44 mL/min. Peptides were detected by continuously measuring the UV absorbance at 214 nm. By running MS scans from 100 to 2000 m/z the mass spectra were collected. MS/MS analysis was performed on the two most intense ions from each scan. A MIX sample consisting of equal volume of all samples analyzed was prepared and this sample was analyzed for each 12 samples. MS data were converted from the Agilent .d format to .mzml files using MSConvert ver. 3.0.6618. All further data analysis was done using R 3.1.3. Peptides were identified from MS/MS spectra using R package ‘MSGFplus’ version 1.05. Search database for peptide identification were limited to the bovine milk proteins: αs1-casein, αs2-casein, β-casein, κ-casein, β-lactoglobulin, α-lactalbumin, lactoperoixdase and lactoferrin. Serine phosphorylation and methionine oxidation were included as variable modifications. R package ‘xcms’ v. 1.42.0 was used for detecting and grouping peaks across samples in a sample set according to Smith et al. (2006). Massifquant method was used for peak detection and grouping of peaks was based on the density method. Identity was assigned to grouped peaks resulting in quantitative tables of identified peptides including β-casein 193-209.

Statistical Analysis of the Positional and Mutational Effects on β-Casein Cleavage

A statistical machine-learning approach and PCA-based analysis was used to determine the effects of all single mutations present in the variants of multi-substitution libraries 1-3, 4 and 6 on cleavage of β-casein at position 192/193.

Results

Multi-Substitution Library 1

Variants of camel chymosin, each having multiple substitutions compared to wild type, were generated and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (mature polypeptide of SEQ ID NO:2), except for the variations mentioned in the table. Both bovine and camel chymosin were included as references.

Clotting activities were determined using the μIMCU method.

TABLE 1 Cleavage of β-casein at position 192/193 of camel chymosin variants 1- 95. Numbers are given in % cleavage of wild type camel chymosin (CHY-MAX M). variant mutations β(193-209) CHY-MAX 795 CHY-MAX M 100 1 I96L G163E V221M 80 2 Y127F R145Q Q188E 172 3 Y21S L166V L253I 110 4 N50K T186S Y307F 109 5 G70N S277N R316L 192 6 I200V Y268F S271P R316L 140 7 M157L T186S I200V S273Y 276 8 D98V G251D M256L V259I 136 9 R67Q H76Q S132A V248I S271P 41 10 Y21S D98V V221K T239S R316L 216 11 V136I T186S V221K I263L S277N 246 12 N50K L222I S255Y 136 14 R67Q V221M M256L 126 15 G70D L166V V317L 1127 16 R67Q L130I M157L 48 17 Y21S R61S H146R 174 18 V136I V221M L222I S226T 67 19 S132A R254S V259I Y307F 54 20 Y21S H76Q Y307F V317L 123 21 D158S L166V V248I F223V G251D 307 22 G70D S74F D158S R254S S277N 195 23 N50K D59N M157L M256L G289S 124 24 M142I V221K T284S 266 25 R61S R67Q K231N 135 26 V32L I96L S277N 25 27 V183I G251W M256L 134 28 M157L T239S D279E 164 29 V248I S226T E294Q 128 30 S74F L166V T186S V203A 101 32 R67Q Y127F V221K G251W 232 33 L130I M142I I200V V259I E294Q 87 34 G70D I96L I200V D267M D279E 161 35 G70N K231N S273Y T284S G289S 174 36 V32L G70N M142I 1024 37 V203A S273Y L295K 115 38 S74F G244D S271P 122 39 L130I G163E Y307F 51 40 R61S L166V T239S 85 41 R254S D279E L295K 999 42 L130I T239S S277N L295K 68 43 G70D V183I Q188E G289S 198 44 R61S G163E M256L S277N 192 46 D98V H146R V203A I263L S271P 85 47 S132A V221M S255Y S273Y V317L 19 48 H76Q L222I G251W 60 49 V221K V248I S255Y 158 50 H76Q K231N G244D 68 51 Y127F S132A D158S 35 52 D59N S271P T284S 119 53 G70D T186S L253I 110 54 R61Q V221K K231N D267M 198 55 V221M V248I L253I L295K 73 56 V183I V248I G244D T284S 102 57 D59N Y127F L166V V183I S255Y 130 58 N50K R61S Y127F G244D G251D 720 59 I96L F223V G244D R254S M256L 903 60 V32L R61Q H146R 22 61 H146R D158S S273Y 949 62 R61Q M142I G289S 182 63 S74F V259I Y268F 971 64 G70N D98V V136I 861 65 D59N V203A R254S 112 66 T239S I263L D267M T284S 124 67 I96L M142I R145Q H146R 780 68 V32L E294Q R316L V317L 27 69 V32L G163E T186S Q188E L295K 752 70 R61Q V136I Y268F T284S Y307F 795 71 S132A Q188E F223V 627 72 H76Q I96L D158S 89 73 V136I R145Q G251D 127 74 R61Q D98V V317L 174 75 Y21S D59N I263L 135 76 I200V G251D G289S 725 77 D98V M157L V183I 84 78 S226T G244D I263L G289S 51 79 Q188E G251D S271P D279E 160 80 N50K D158S V203A E294Q 682 81 V203A V248I G251W L253I Y268F 152 82 R61S V183I L222I L253I D267M 100 84 G70D L130I Y268F 49 85 Y127F D267M E294Q 163 88 F223V V248I I263L 248 89 G70N R254S S255Y Y268F 105 90 D59N V248I L222I V248I 90 91 F223V G251W S273Y D279E 352 92 R67Q G70N H146R Q188E S226T 84 93 S74F H76Q M142I M157L G163E 99 94 R61Q S226T T239S V248I G251W 53 95 V32L L130I R145Q L222I D279E 5

In Table 1 are shown camel chymosin variants with data on cleavage of β-casein at position 192/193. Since all enzyme variants were used at a normalized concentration of 0.05 IMCU/mL in the experiments, low β-casein cleavage indicates high specificity of the respective variant for κ-casein 104/105 over β-casein 192/193 cleavage, rather than low general enzymatic activity.

Variants with half or less than wild type proteolytic activity on β-casein are highlighted in bold (variants 9, 16, 26, 39, 47, 51, 60, 68, 78, 84, 95). In those, mutations V32L, L130I, and S132A are overrepresented, compared to the mutational pattern present in the entire variant set shown. Four out of six variants with mutation V32L, four out of six variants with mutation L130I, and three out of five variants with mutation S132A show β-casein 192/193 cleavage equal or less than 50% of wild type camel chymosin.

In the three-dimensional structure of camel chymosin, position V32 is interacting with the P1 residue of the substrate peptide sequence (FIG. 2), while positions L130 and S132 are interacting with P5″ (L130) as well as P2′ and P6′ (S132), respectively (FIG. 3; references 5-10). The location of the three positions in the chymosin substrate binding site suggests that mutations V32L, L130I, and S132A cause lower β-casein 192/193 cleavage and, thus, lower generation of the β-casein fragment β(193-209) at constant coagulant strengths by direct interaction with κ- and β-casein. Variant 95, which is showing the lowest β-casein 192/193 cleavage throughout the variant set, contains both mutations V32L and L130I. This suggests additivity of the mutational effects on casein substrate specificity.

Multi-Substitution Library 2

Another set of camel chymosin variants, each having multiple substitutions compared to wild type, were generated and analyzed as described. All variants have an amino acid sequence identical to camel chymosin, except for the variations mentioned in the table. Both bovine and camel chymosin were included as references. Clotting activities were determined using the REMCAT method.

TABLE 2 Cleavage of β-casein at position 192/193 of camel chymosin variants 96- 143. Numbers are given in % cleavage of wild type camel chymosin (CHY-MAX M). variant mutations β(193-209) CHY-MAX 488 CHY-MAX M 100 96 D59N L222I G251D E83S Q162S 70 97 D59N L222I G251W F17Y Y21S 85 98 D59N L222I G251D H76Q S164G 29 99 D59N L222I G251D K62Q M165E 94 100 D59N L222I G251D Q162S V155F 74 101 D59N L222I G251D H76Q V155F 284 102 D59N L222I G251D S273Y L166V 75 103 D59N L222I G251D Y268F V198I 72 104 D59N L222I G251D S273Y F66Y 64 105 D59N L222I G251D M165E L166V 70 106 D59N L222I G251D H76Q M165E 63 107 D59N L222I G251D F17Y S273Y 76 108 D59N L222I G251D L166V I45V 84 109 D59N L222I G251W L180I T284S 84 110 D59N L222I G251D V32L L12M T284S 20 111 D59N L222I G251D Y21S L166V 61 112 D59N L222I G251D V155F E262T V32L 16 113 D59N L222I G251D L105E S164G 52 114 D59N L222I G251W S154A V203A 105 115 D59N L222I G251D Q162S L166V 233 116 D59N L222I G251W K19T R266I 100 117 D59N L222I G251W I303L I45V 103 119 D59N L222I G251D Y21S L215V L105E 74 120 D59N L222I G251D I96L T177S K321P 86 121 D59N L222I G251D F17Y T284S V203A 84 122 D59N L222I G251D V32L K321P V260T 13 123 D59N L222I G251D V198I V32L E83S 82 124 D59N L222I G251D I96L V203A V309I 54 125 D59N L222I G251D Y268F L215V V32L 11 126 D59N L222I G251D H76Q L105E V260T 41 127 D59N L222I G251D Y21S H76Q Y268F 30 128 D59N L222I G251D Y21S I45V F223A 295 129 D59N L222I G251D V198I V203A K321P 109 131 D59N L222I G251D S164G R266V I96L 39 132 D59N L222I G251D H181N F66Y V32L 10 133 D59N L222I G251D H181N R266I D267Q 91 134 D59N L222I G251W K62Q V309I 103 135 D59N L222I G251D Y268F L12M D267Q 64 136 D59N L222I G251D L166V E262T T177S 97 137 D59N L222I G251D S273Y T284S D267Q 107 138 D59N L222I G251D F66Y Q288E I96L 51 139 D59N L222I G251D V203A R266V F223A 48 140 D59N L222I G251D I303L S154A V260T 59 141 D59N L222I G251D Y21S T284S I96L 48 142 D59N L222I G251D Q288E K19T T177S 45 143 D59N L222I G251D K62Q Y268F K19T 55

In Tab. 2 are shown camel chymosin variants with data on cleavage of β-casein at position 192/193. Since all enzyme variants were used at a normalized concentration of 0.05 IMCU/mL in the experiments, low β-casein cleavage indicates high specificity of the respective variant for κ-casein 104/105 over β-casein 192/193 cleavage, rather than low general enzymatic activity.

Variants with less than 25% wild type proteolytic activity on β-casein are highlighted in bold (variants 110, 112, 122, 125, 132). In those, mutation V32L is overrepresented, compared to the mutational pattern present in the entire variant set shown. Five out of six variants with mutation V32L show β-casein 192/193 cleavage equal or less than 25% of wild type camel chymosin. These results support the findings and conclusions of the previous variant set.

Multi-Substitution Library 3

A third set of camel chymosin variants, each having multiple substitutions compared to wild type, were generated and analyzed as described. All variants have an amino acid sequence identical to camel chymosin, except for the variations mentioned in the table. Both bovine and camel chymosin were included as references. Clotting activities were determined using the μIMCU method.

TABLE 3 Cleavage of β-casein at position 192/193 of camel chymosin variants 144-179. Numbers are given in % cleavage of β-casein of wild type camel chymosin (CHY-MAX M). Var. mutations β (193-209) CHY-MAX 791 CHY-MAX M 100 144 L12M Y21S D59N H76Q M165E V198I L222I G251D Q288E 20 146 L12M Y21S D59N H76Q M165E L222I G251W S273Y 25 147 L12M D59N H76Q M165E V198I L222I G251D S273Y K321P 27 148 L12M D59N H76Q S154A M165E V203A L222I G251D V309I 23 149 L12M D59N H76Q D98V L222I 31 150 L12M K19T V32L D59N H76Q D144Q M165E L222I G251D 6 151 L12M Y21S D59N H76Q M165E V203A L222I G251D E262T 26 152 L12M V51L H76Q M165E G251D 41 153 L12M D59N F66Y H76Q M165E L180I L222I G251D V309I 29 154 L12M D59N H76Q S154A M165E L222I G251W Q288E 25 155 L12M D59N H76Q D98V M165E L222I G251D E262T Q288E 23 156 L12M V51L D59N H76Q L166V L222I G251D 17 157 L12M D59N H76Q D144Q M165E V203A L222I 30 158 L12M D59N D144Q M165E L166V L222I G251D 38 159 L12M K19T D59N H76Q S154A M165E V198I L222I G251D 16 160 L12M H76Q D98V M165E L222I G251W 36 161 L12M V32L D59N H76Q M165E L180I V198I L222I G251D 8 162 L12M D59N H76Q S154A M165E S273Y 46 164 L12M V51L D59N F66Y H76Q M165E V203A L222I G251W 36 165 L12M V32L H76Q M165E L222I E262T 8 166 L12M N50D D59N H76Q M165E G251W E262T 40 168 V51L D59N H76Q M165E L180I L222I G251D E262T 36 169 L12M D59N H76Q M165E G251D Q288E V309I K321P 39 172 L12M N50D D59N V203A L222I G251D 40 173 L12M D59N H76Q L180I L222I G251W K321P 25 174 L12M Y21S D59N M165E L222I K321P 48 176 D59N H76Q M165E L166V V198I L222I 63 178 L12M K19T N50D D59N H76Q M165E L222I Q288E 30 179 L12M Y21S N50D D59N F66Y H76Q D144Q M165E L222I G251D 36

In Tab. 3 are shown camel chymosin variants with data on cleavage of β-casein at position 192/193. Since all enzyme variants were used at a normalized concentration of 0.05 IMCU/mL in the experiments, low β-casein cleavage indicates high specificity of the respective variant for κ-casein 104/105 over β-casein 192/193 cleavage, rather than low general enzymatic activity.

Variants with less than 10% wild type proteolytic activity on β-casein are highlighted in bold (variants 150, 161, 165). In those, mutation V32L is overrepresented, compared to the mutational pattern present in the entire variant set shown. All three variants with mutation V32L show β-casein 192/193 cleavage less than 10% of wild type camel chymosin.

Only one variant from this variant set (variant 176) is showing higher than 50% β-casein 192/193 cleavage compared to wild type camel chymosin. This is also the only variant from this set lacking mutation L12M.

Position L12 is located in the sequence stretch close to the N-terminus of camel chymosin that is bound in the substrate binding cleft of the enzyme (FIG. 4). It has been described that in camel chymosin the N-terminal sequence is blocking the substrate binding cleft of the enzyme when no substrate is bound (reference 5). Casein substrate molecules need to replace this N-terminal sequence in order to bind to the active site and subsequently get cleaved. Mutations in chymosin that are stabilizing this inactive form of the enzyme can consequently reduce substrate binding and, thus, affect casein cleavage specificity. We conclude this mode of action for mutation L12M. In the three-dimensional structure of camel chymosin, positions L12 and V32 are in direct contact with each other. In addition to its direct impact on β-casein binding, V32L might as well stabilize the inactive form of the enzyme. Since variants containing both mutations (150, 161, 165) show lowest β-casein 192/193 cleavage amongst all variants of this set, their impact on casein substrate specificity seems to be additive.

Mutational Analysis of Multi-Substitution Libraries 1-3

A statistical analysis of the positional and mutational effects on β-casein cleavage was performed based on the proteolytic data of libraries 1-3. The most beneficial mutations for decreased β-casein cleavage are shown in table 4.

TABLE 4 Mutational contributions (mean) to reduced β-casein 192/193 cleavage and standard deviations (sd) based on statistical analysis. mutation mean sd L130I 2.43E−01 4.32E−02 S132A 1.96E−01 6.49E−02 V32L 1.87E−01 5.95E−02 S226T 1.67E−01 4.04E−02 R266V 1.54E−01 4.35E−02 L12M 1.45E−01 2.23E−02 V221M 1.35E−01 3.48E−02 S255Y 1.09E−01 4.62E−02 S277N 1.05E−01 4.15E−02 L222I 9.64E−02 2.22E−02 L253I 8.78E−02 3.13E−02 M157L 8.67E−02 3.50E−02 V260T 8.33E−02 3.76E−02 S271P 8.04E−02 3.21E−02 H76Q 7.68E−02 2.67E−02 K19T 6.76E−02 2.57E−02 V183I 6.64E−02 3.05E−02 S164G 6.51E−02 2.15E−02 I263L 6.37E−02 2.77E−02 V51L 6.25E−02 3.01E−02 T239S 6.25E−02 3.32E−02 E262T 6.03E−02 2.61E−02 K231N 5.78E−02 2.93E−02 R316L 5.22E−02 4.55E−02 I96L 4.81E−02 3.22E−02

Based on the obtained results it is concluded that mutations shown in table 4 reduce β-casein 192/193 cleavage, with the above described mutations L130I, S132A, V32L, and L12M being amongst the mutations with the strongest impact (highlighted in bold in table 4).

Since the mutations shown in table 4 cause less generation of the C-terminal fragment of β-casein, β(193-209), they represent preferred mutations in chymosin variants for making cheese with less bitter taste due to reduced cleavage of β-casein.

Multi-Substitution Library 4

Another set of camel chymosin variants, each having multiple substitutions compared to wild type, were generated and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (mature polypeptide of SEQ ID NO:2), except for the variations mentioned in the table. Camel chymosin (CHY-MAX M) is included as reference.

Clotting activities were determined using the μIMCU method.

TABLE 5 Cleavage of β-casein at position 192/193 of camel chymosin variants 180-222. Numbers are given in % cleavage of β-casein of wild type camel chymosin(CHY-MAX). variant mutations β(193-209) CHY-MAX M 100 180 H76Q S132A S164G L222I N249D G251D 14 181 Y21S D59N H76Q S164G L166V N249D G251D S273Y 53 182 D59N H76Q S164G L222I R242E S273Y V309I 35 183 D59N H76Q L130I L166V L222I N249D G251D S273Y 22 184 Y21S D59N S164G L222I R242E G251D S273Y V309I 42 185 K19T Y21S D59N H76Q S132A S164G L222I G251D S273Y 12 186 D59N H76Q I96L L130I S164G L222I R242E G251D 9 187 H76Q S164G L166V L222I S226T S273Y 26 188 K19T D59N I96L S164G L222I G251D 33 189 Y21S H76Q S164G L222I R242E G251D S273Y 23 190 H76Q I96L S164G L222I R242E G251D S273Y 23 191 H76Q S164G L222I N249D G251D S273Y V309I 31 192 K19T D59N H76Q S164G L222I N249D S273Y 20 193 Y21S D59N H76Q S164G L222I S226T G251D S273Y V309I 21 194 H76Q S164G L166V L222I R242E G251D S273Y 21 195 D59N H76Q I96L S164G L222I S226T N249D G251D S273Y 19 196 D59N H76Q L130I S164G L166V L222I G251D S273Y V309I 14 197 D59N S132A S164G L222I R242E N249D G251D S273Y 12 198 H76Q I96L S164G G251D S273Y V309I 39 199 D59N H76Q L130I S164G G251D V309I 13 200 K19T D59N S164G L166V L222I S226T G251D S273Y 28 201 D59N H76Q I96L S132A S164G L222I S226T G251D S273Y 8 202 K19T D59N H76Q I96L S164G L166V L222I G251D S273Y 17 203 K19T D59N H76Q L130I S164G L222I S226T G251D S273Y 11 204 K19T D59N H76Q S132A L222I G251D S273Y V309I 18 205 H76Q L130I L222I S226T G251D S273Y 14 206 K19T Y21S D59N H76Q L130I S164G L222I S273Y 15 207 Y21S D59N H76Q I96L S164G L222I N249D G251D S273Y 28 208 K19T D59N H76Q S164G R242E N249D G251D S273Y 24 209 D59N H76Q S164G L222I S226T R242E 15 210 D59N H76Q I96L S132A S164G L166V L222I G251D S273Y 11 211 D59N H76Q S132A S164G L166V S273Y 17 212 Y21S D59N S164G L222I S226T N249D G251D S273Y 33 213 D59N H76Q L130I S132A S164G L222I R242E G251D S273Y 7 214 D59N H76Q S164G L166V L222I N249D G251D S273Y V309I 32 215 D59N H76Q I96L S164G L222I S226T G251D S273Y V309I 18 216 K19T D59N H76Q L166V L222I R242E G251D S273Y 43 217 Y21S D59N H76Q I96L L222I S273Y 56 218 D59N H76Q I96L L130I S164G L222I N249D G251D S273Y 14 219 L130I S164G L222I S273Y 19 220 K19T Y21S H76Q S164G L222I G251D S273Y 36 221 Y21S D59N H76Q L130I S132A S164G L222I G251D S273Y 11 222 D59N H76Q S226T R242E G251D S273Y 54

In table 5 are shown camel chymosin variants with data on cleavage of β-casein 192/193. All variants reveal between 44% and 93% reduced proteolytic activity compared to wild type camel chymosin.

Mutational Analysis of Multi-Substitution Library 4

A statistical analysis of the positional and mutational effects on β-casein cleavage was performed based on the proteolytic data of library 4 variants. The most beneficial mutations for decreased β-casein cleavage are shown in table 6.

TABLE 6 Mutational contributions (mean) to reduced β-casein 192/193 cleavage and standard deviations (sd) based on statistical analysis. mutation mean sd S132A 1.10E+00 1.17E−01 L130I 1.07E+00 1.14E−01 S164G 1.02E+00 1.29E−01 L222I 6.50E−01 1.31E−01 S226T 6.49E−01 1.05E−01 H76Q 6.37E−01 1.06E−01 R242E 4.45E−01 1.24E−01 K19T 4.18E−01 1.13E−01 N249D 3.38E−01 1.03E−01 L166V 3.07E−01 9.43E−02 I96L 2.93E−01 1.02E−01 V309I 2.08E−01 1.23E−01

Based on the obtained results it is concluded that mutations shown in table 6 reduce β-casein 192/193 cleavage.

Since these mutations cause less generation of the C-terminal fragment of β-casein, β(193-209), they represent preferred mutations in chymosin variants for making cheese with less bitter taste due to reduced cleavage of β-casein.

Multi-Substitution Library 5

Another set of camel chymosin variants, each having multiple substitutions compared to wild type, were generated and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (mature polypeptide of SEQ ID NO:2), except for the variations mentioned in the table. Camel chymosin (CHY-MAX M) is included as reference.

Clotting activities were determined using the REMCAT method.

TABLE 7 Cleavage of β-casein at position 192/193 of camel chymosin variants 223-269. Numbers are given in % cleavage of β-casein of wild type camel chymosin (CHY-MAX M). variant mutations β(193-209) CHY-MAX M 100 223 K19T D59N I96L S164G L222I G251D 46 224 Y11I K19T D59N I96V L222I R242D G251D 47 225 K19S D59N I96V S164G G251D 59 226 K19S I96L S164G L166V L222I R242E 20 227 K19T D59N I96L S164G L166V L222I R242D G251D L253I 25 228 D59N I96L S164G L222I R242E L253I I263L 27 229 K19T D59N E83T I96L L222I G251D I263L 65 230 Y11I K19T D59N S164G L222I G251D I263V 28 231 K19T D59N I96L S164G L166I G251D L253V 44 232 K19T I96V S164G L222I N249D G251D L253I 50 233 K19T I96L L222I R242E L253I 54 234 K19T E83S I96L S164G L222I R242E G251D L253I 21 235 D59N E83T I96L S164N L222V G251D 119 236 K19S D59N I96L S164G L222I R242E N249E G251D 31 237 K19T I96L S164G L166V L222I N249D I263L 32 238 D59N I96L L166V L222I R242E G251D 44 239 K19T D59N E83T S164G L166V L222I R242D G251D 31 240 Y11I K19T D59N E83S I96L S164G L222I N249D 24 241 K19T E83T I96L S164G L222I R242E L253V 24 242 K19T D59N I96L S164G L166I L222I R242E N249D 28 243 Y11V K19T D59N I96L S164G L166V L222I R242E G251D L253I 17 244 K19T I96L S164N L222I R242E I263L 72 245 Y11V D59N I96L S164G L222I G251D L253V 30 246 K19T D59N I96V S164G L166V L222I R242E I263L 25 247 Y11V K19T D59N I96L S164N L166I L222I G251D 67 248 K19T I96L S164G L166V L222I R242E N249D G251D I263V 33 249 K19T I96L S164G R242E L253I 42 250 K19S D59N E83S I96L S164N L222I G251D 84 251 K19T D59N I96L S164G L222V N249E G251D I263V 40 252 K19T D59N I96L S164G L222I N249E G251D L253V I263L 33 253 Y11I K19T I96L S164G L222V R242E G251D 29 254 I96L S164G L222I R242E N249D G251D I263L 29 255 K19T D59N I96L S164G L166I L222I R242D G251D I263V 28 256 K19T D59N I96L S164G L222V R242E N249D L253I 42 257 H76Q I96L S164G L222I R242E G251D S273Y 23 258 K19T E83S I96L S164G L222I R242E N249D G251D L253I 22 259 I96L S164G L166V L222I R242E N249D I263L 34 260 Y11V K19T E83S I96L S164G L166V L222I R242E G251D 20 261 Y11V K19T I96L S164G L166V L222I R242E 30 262 Y11V E83S I96L S164G L222I R242E G251D L253I I263L 21 263 Y11V I96L S164G L222I R242E N249D L253I I263L 23 264 K19T I96L S164G L166V L222I R242E N249D I263L 35 265 Y11V E83S I96L S164G L222I R242E L253I I263L 24 266 K19T E83S I96L S164G L166V L222I R242E N249D G251D L253I 26 267 I96L S164G L222I R242E G251D S274Y 42 268 H76Q I96L S164G L222I R242E G251D 25 269 I96L S164G L222I R242E G251D 41

In Table 7 are shown camel chymosin variants with data on cleavage of β-casein 192/193. Out of 47 variants, 46 reveal between 16% and 83% reduced proteolytic activity compared to wild type camel chymosin.

Mutational Analysis of Multi-Substitution Library 5

A statistical analysis of the positional and mutational effects on β-casein cleavage was performed based on the proteolytic data of library 5 variants. The most beneficial mutations for decreased β-casein cleavage are shown in table 8.

TABLE 8 Mutational contributions (mean) to reduced β-casein 192/193 cleavage and standard deviations (sd) based on statistical analysis. mutation mean sd S164G 5.08E−01 2.40E−02 R242E 2.76E−01 2.94E−02 Y11V 2.70E−01 2.91E−02 L222I 2.22E−01 2.76E−02 E83S 2.07E−01 3.61E−02 Y11I 2.04E−01 2.91E−02 H76Q 1.68E−01 3.13E−02 D59N 1.24E−01 3.03E−02 L166V 1.06E−01 3.75E−02 R242D 1.01E−01 2.09E−02 L253I 7.93E−02 3.26E−02 L253V 6.87E−02 2.82E−02 K19S 4.62E−02 4.19E−02 I96L 4.08E−02 2.64E−02 I263V 3.56E−02 2.86E−02 E83T 3.21E−02 2.95E−02

Based on the obtained results it is concluded that mutations shown in table 8 reduce β-casein 192/193 cleavage.

Since these mutations cause less generation of the C-terminal fragment of β-casein, β(193-209), they represent preferred mutations in chymosin variants for making cheese with less bitter taste due to reduced cleavage of β-casein.

Structure-Based Variations in Camel Chymosin

Variants of camel chymosin (SEQ ID NO:2) were made with amino acid changes in positions determined by protein structural analysis (Tab. 9). Mutations N100Q and N291Q were introduced into both N-glycosylation sites of these variants and the reference camel chymosin (CamUGly) to yield non-glycosylated, homogeneous protein samples.

Clotting activities were determined using the μIMCU method.

TABLE 9 Cleavage of β-casein at position 192/193 of camel chymosin variants 270-308. Numbers are given in % cleavage of non-glycosylated camel chymosin (CamUGly). variant mutations β(193-209) CamUGly N100Q N291Q 100 270 V32L N100Q N291Q 28 271 V221K N100Q N291Q 143 272 D290E N100Q N291Q 60 273 V136I N100Q N291Q 111 274 E240Q N100Q N291Q 109 275 R242Q N100Q N291Q 74 276 G289S N100Q N291Q 51 277 N292H N100Q N291Q 164 278 L295K N100Q N291Q 131 279 V136E N100Q N291Q 99 280 D290L N100Q N291Q 58 281 F119Y N100Q N291Q 107 282 Q280E N100Q N291Q 85 283 F282E N100Q N291Q 79 285 R254S N100Q N291Q 78 286 R242E N100Q N291Q 89 288 V203R N100Q N291Q 115 289 N249R N100Q N291Q 90 290 H56K N100Q N291Q 140 291 S74D N100Q N291Q 101 292 A131D N100Q N291Q 230 293 Y190A N100Q N291Q 28 294 I297A N100Q N291Q 185 295 H76Q N100Q N291Q 48 296 S273Y N100Q N291Q 58 297 K19T N100Q N291Q 66 298 D59N N100Q N291Q 60 299 L222I N100Q N291Q 54 300 V309I N100Q N291Q 70 301 I96L N100Q N291Q 75 302 Y21S N100Q N291Q 67 303 L130I N100Q N291Q 29 304 S132A N100Q N291Q 28 305 S226T N100Q N291Q 44 306 G251D N100Q N291Q 88 307 Y243E N100Q N291Q 62 308 S273D N100Q N291Q 66

Based on the results shown in table 9 it is concluded that mutations K19T, Y21S, V32L, D59N, H76Q, I96L, L130I, S132A, Y190A, L222I, S226T, D290E, D290L, R242E, R242Q, Y243E, G251D, R254S, S273D, S273Y, Q280E, F282E, G289S, and V309I reduce cleavage of β-casein 192/193 by more than 10%.

Since these mutations cause less generation of the C-terminal fragment of β-casein, β(193-209), they represent preferred mutations in chymosin variants for making cheese with less bitter taste due to reduced cleavage of β-casein.

Ten out of 24 variants with decreased cleavage of β-casein 192/193 shown in table 9 bear mutations (V32L, H76Q, L130I, S132A, Y190A, L222I, S226T, G289S, D290E, D290L) within or in structural proximity to the substrate binding cleft (FIG. 5), suggesting a direct impact of these mutations on β-casein binding.

Nine out of 24 variants with decreased cleavage of β-casein 192/193 shown in table 9 bear mutations (R242E, R242Q, Y243E, G251D, R254S, S273D, S273Y, Q280E, F282E) in a distinct region on the protein surface that is located in proximity to the binding cleft as seen in FIG. 6. This region has been suggested to support binding of the κ-casein substrate by interacting with its positively charged sequence Arg96 to His102 (references 5, 16-18) in positions P10 to P4 (reference 10). The introduced mutations may strengthen these interactions by reducing the net charge of this region on the protein surface. Increased binding of κ-casein will ultimately inhibit binding and hydrolysis of other substrates such as β-casein. The results show that single amino acid substitutions in this region can increase C/P significantly.

Negative Charge Combinations in Camel Chymosin

More variants of camel chymosin (SEQ ID NO:2) were made with combinations of mutations that introduce negative charges into the surface region described above (R242E, Y243E, G251D, N252D, R254E, S273D, Q280E). Mutations N100Q and N291Q were introduced into both N-glycosylation sites of these variants and the reference camel chymosin (CamUGly) to yield non-glycosylated, homogeneous protein samples (Tab. 10).

Clotting activities were determined using the pIMCU method.

TABLE 10 Cleavage of β-casein at position 192/193 of camel chymosin variants 309-323. Numbers are given in % cleavage of non-glycosylated camel chymosin (CamUGly). variant mutations β(193-209) CamUGly N100Q N291Q 100 309 R242E Q280E N100Q N291Q 50 310 R242E N252D N100Q N291Q 65 311 N252D Q280E N100Q N291Q 61 312 Y243E Q280E N100Q N291Q 59 313 Y243E N252D N100Q N291Q 62 314 R254E Q280E N100Q N291Q 66 315 S273D Q280E N100Q N291Q 85 316 R242E G251D N100Q N291Q 92 317 R242E G251D Q280E N100Q N291Q 73 318 R242E S273D Q280E N100Q N291Q 81 319 N252D S273D Q280E N100Q N291Q 89 320 G251D S273D Q280E N100Q N291Q 96 321 R242E R254E Q280E N100Q N291Q 92 322 R242E R254E S273D Q280E N100Q N291Q 72 323 Y243E R254E S273D N100Q N291Q 70

All variants shown in table 10 reveal decreased β-casein cleavage compared to non-glycosylated camel chymosin. It is concluded that the inhibition of β-casein cleavage by introducing negative charges into the P10-P4 interacting region on the chymosin structure can be further enhanced by combinations of the respective mutations.

Structure-Based Variations in Bovine Chymosin

Variants of bovine chymosin (SEQ ID NO:1) were made with amino acid changes in positions determined by protein structural analysis (Tab. 11). Mutations N252Q and N291Q were introduced into both N-glycosylation sites of these variants and the reference bovine chymosin (BovUGly) to yield non-glycosylated homogeneous protein samples.

Clotting activities were determined using the μIMCU method.

TABLE 11 Cleavage of β-casein at position 192/193 of bovine chymosin variants 325-346. Numbers are given in % cleavage of non-glycosylated bovine chymosin (BovUGly). variant mutations β(193-209) BovUGly N252Q N291Q 100 325 V223F N252Q N291Q 171 326 E290D N252Q N291Q 157 327 A117S N252Q N291Q 119 328 I136V N252Q N291Q 93 329 Q242R N252Q N291Q 146 330 Q278K N252Q N291Q 139 331 S289G N252Q N291Q 145 333 Q294E N252Q N291Q 155 335 D249N N252Q N291Q 171 336 D251G N252Q N291Q 143 337 G244D N252Q N291Q 103 338 Q56H N252Q N291Q 125 339 L32I N252Q N291Q 121 340 K71E N252Q N291Q 133 341 P72T N252Q N291Q 106 342 Q83T N252Q N291Q 122 343 V113F N252Q N291Q 159 344 E133S N252Q N291Q 141 345 Y134G N252Q N291Q 105 346 K71A N252Q N291Q 123

Except I136V, all mutations caused increased cleavage of β-casein 192/193 in the variants shown in table 11. Notably, while substitutions I136V, Q242R, D251G, S289G, and E290D increased β-casein cleavage of bovine chymosin, decreased β-casein cleavage was observed by the respective reverse mutations V136I, R242Q, G251D, G289S, and D290E in camel chymosin (Tab. 9). A similar effect is seen in position 32. While V32L caused decreased β-casein cleavage of camel chymosin, mutation of L32 to I—a β branched hydrophobic amino acid with structural similarity to V—resulted in increased β-casein cleavage of bovine chymosin. This demonstrates that these amino acid changes exert similar effects on chymosin specificity across species.

Variations of the Camel Chymosin N-terminus

Variants of camel chymosin (SEQ ID NO:2) were made with amino acid changes in positions determined by protein structural analysis of the molecular interactions of the N-terminal sequence Y11-D13 within the substrate binding cleft (Tab. 12). Mutations N100Q and N291Q were introduced into both N-glycosylation sites of these variants and the reference camel chymosin (CamUGly) to yield non-glycosylated, homogeneous protein samples.

Clotting activities were determined using the μIMCU method.

TABLE 12 Cleavage of β-casein at position 192/193 of camel chymosin variants 347-366. Numbers are given in % cleavage of non-glycosylated camel chymosin (CamUGly). variant mutations β(193-209) CamUGly N100Q N291Q 100 347 Y11H N100Q N291Q 109 348 Y11K N100Q N291Q 126 349 Y11R N100Q N291Q 100 350 Y11H D290E N100Q N291Q 50 351 Y11R D290E N100Q N291Q 40 352 Y11F N100Q N291Q 105 353 Y11I N100Q N291Q 98 354 Y11L N100Q N291Q 93 356 L12F N100Q N291Q 102 357 L12I N100Q N291Q 91 359 D13N N100Q N291Q 127 360 D13Q N100Q N291Q 109 361 D13S N100Q N291Q 131 362 D13T N100Q N291Q 155 363 D13F N100Q N291Q 108 364 D13L N100Q N291Q 120 365 D13V N100Q N291Q 136 366 D13Y N100Q N291Q 124

Analysis of the camel chymosin structure guided variations in the N-terminal sequence Y11-D13 as well as in position D290, a potential interaction partner of Y11 (FIG. 7). Since casein substrates compete with the N-terminal chymosin sequence for binding within the binding cleft, amino acid substitutions that change interactions between binding cleft and the motif Y11-D13 are expected to impact enzymatic activity toward various casein substrates and, thus, cleavage of β-casein 192/193. The results of the respective variants 347-366 show strong variation of β-casein cleavage (Tab. 12). Notably, variants 353 and 355—both bearing mutation D290E—reveal decreased β-casein cleavage.

Multi-Substitution Library 6

Another set of camel chymosin variants, each having multiple substitutions compared to wild type, were generated and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (mature polypeptide of SEQ ID NO:2), except for the variations mentioned in the table. Camel chymosin (CHY-MAX M) is included as reference.

Clotting activities were determined using the pIMCU method.

TABLE 13 Cleavage of β-casein at position 192/193 of camel chymosin variants 367- 416. Numbers are given in % cleavage of wild type camel chymosin (CHY-MAX M). variant mutations β(193-209) CHY-MAX M 100 367 R67Q N100Q L130I M157L V248I N291Q 44 368 N100Q L130I S132A M157L K231N 24 369 R67Q I96L L130I M157L L222I M256L 13 370 R67Q L130I S132A M157L R242E V248I 17 371 R67Q N100Q M157L R242E M256L 69 372 R67Q G70D M157L R242E V248I 60 373 V32L R67Q M157L L222I R242E 9 374 Y11V R67Q M157L V248I M256L 72 375 R67Q V136I M157L L222I V248I 26 376 L130I M157L V248I M256L N291Q 28 377 R67Q I96L L130I M157L K231N R242E 20 378 V32L R67Q L130I M157L L222I K231N 5 379 L130I V136I M157L L222I N292H 22 380 R67Q G70D M157L L222I N291Q 81 381 V32L R67Q L130I K231N N292H 6 382 Y11V R67Q N100Q L130I V136I M157L 31 383 R67Q L130I L222I R242E M256L 14 384 R67Q M157L L222I V248I N292H 62 385 V32L R67Q M157L M256L N291Q 12 386 R67Q L130I S132A M157L L222I N292H 10 387 R67Q N100Q L130I M157L K231N N291Q 47 388 R67Q L130I K231N V248I N291Q 43 389 Y11V R67Q L130I M157L L222I K231N 13 390 I45V L130I M157L K231N R242E 15 391 V32L R67Q V136I M157L N291Q 17 392 R67Q N100Q L130I D158S V248I 32 393 I45V R67Q L130I M157L L222I K231N 14 394 V32L R67Q L130I S132A M157L V248I 3 395 Y11V R67Q L130I M157L N291Q N292H 30 396 R67Q N100Q L130I M157L L222I K231N 20 397 I45V R67Q G70D L130I S132A 18 398 I45V R67Q L130I V248I N292H 39 399 Y11V R67Q L130I M157L L222I R242E 11 400 R67Q N100Q D158S L130I M157L L222I 19 401 R67Q L130I V136I M157L K231N V248I 25 402 I45V R67Q L130I L222I N291Q 24 403 R67Q G70D L130I M157L K231N M256L 23 404 V32L R67Q L130I M157L D158S V248I 5 405 R67Q L130I M157L D158S R242E N291Q 30 406 R67Q L130I M157L D158S K231N N292H 30 407 R67Q L130I V248I M256L N292H 42 408 V32L R67Q I96L L130I M157L V248I 12 409 R67Q I96L N100Q L130I M157L N292H 58 410 V32L R67Q G70D N100Q M157L 15 411 V32L R67Q L130I M157L K231N M256L 32 412 R67Q I96L M157L L222I K231N 72 413 R67Q M157L L222I K231N V248I 70 414 R67Q L130I M157L R242E M256L N292H 23 415 R67Q L222I K231N V248I 77 416 R67Q S132A L222I K231N R242E V248I 13

In Table 13 are shown camel chymosin variants with data on cleavage of β-casein 192/193. All 50 variants reveal between 19% and 97% reduced proteolytic activity compared to wild type camel chymosin.

Mutational Analysis of Multi-Substitution Library 6

A statistical analysis of the positional and mutational effects on β-casein cleavage was performed based on the proteolytic data of library 6 variants. The most beneficial mutations for decreased β-casein cleavage are shown in Table 14.

TABLE 14 Mutational contributions (mean) to reduced β-casein 192/193 cleavage and standard deviations (sd) based on statistical analysis. mutation mean sd V32L 4.19E−01 2.43E−02 L130I 1.92E−01 1.47E−02 S132A 1.88E−01 3.28E−02 L222I 7.39E−02 1.09E−02 M157L 3.28E−02 1.47E−02 D158S 2.50E−02 2.08E−02 R67Q 2.44E−02 1.15E−02 Y11V 2.04E−02 8.41E−03 M256L 2.00E−02 1.12E−02

Based on the obtained results it is concluded that mutations shown in Table 14 reduce β-casein 192/193 cleavage.

Since these mutations cause less generation of the C-terminal fragment of β-casein, β(193-209), they represent preferred mutations in chymosin variants for making cheese with less bitter taste due to reduced cleavage of β-casein.

Another set of camel chymosin variants, each having multiple substitutions compared to the wild type, were generated and analyzed as described above. All variants have an amino acid sequence identical to camel chymosin (mature polypeptide of SEQ ID NO:2), except for the variations mentioned in the table. Camel chymosin (CHY-MAX M) is included as reference.

Clotting activities were determined using the μIMCU method.

TABLE 15 Cleavage of β-casein at position 192/193 (β), specific clotting (C), proteolysis (P) and C/P ratio of camel chymosin variants 417-461. Numbers are given in % of wild type camel chymosin, CHY-MAX M (CMM). variant mutations β (C) (P) C/P CMM 100 100 100 100 417 Y11V K19T D59N S164G L166V L222I R242E N249E G251D 21 132 20 651 418 Y11V K19T D59N I96L S164G L166I L222I R242E N249E G251D 18 114 21 556 419 Y11I K19T D59N I96L S164G L166V L222I R242E N249E G251D 17 108 20 554 420 Y11I K19T D59N I96L S164G L166I L222I R242E G251D 18 98 11 898 421 Y11V K19T D59N I96L L166V L222V R242E N249E G251D L253I 36 132 84 156 422 Y11V K19T D59N I96L S164G L166V R242E 38 105 13 802 423 Y11V K19T D59N I96L S164G L222V R242E G251D 28 89 8 1131 424 Y11V K19T D59N I96L S164G L166I R242E N249E G251D L253I 20 93 8 1111 425 Y11V K19T D59N I96L S164G L166V L222V R242E N249E G251D 21 105 18 572 426 Y11V K19T D59N I96L S164G L166I L222V R242E N249E G251D L253I 16 93 18 512 427 Y11V K19T D59N L166V L222I R242E N249E G251D L253I 33 137 42 323 428 Y11V K19T D59N I96L S164G L166V L222I R242E N249E 20 120 15 803 429 Y11V K19T D59N S164G L166I L222I R242E G251D 25 107 17 630 430 Y11V K19T D59N I96L S164G R242E G251D 25 89 11 801 431 Y11V D59N I96L S164G L166I L222V R242E G251D L253I 23 79 28 283 432 Y11V D59N I96L S164G L166I L222I R242E G251D 16 102 24 432 433 Y11I D59N I96L S164G L166V L222V R242E G251D L253I 17 97 25 392 434 Y11V K19T D59N I96L S164G L222I R242E N249E G251D 17 99 33 301 435 Y11V K19T D59N I96L S164G L166I L222V R242E G251D 25 88 17 514 436 Y11V K19T D59N I96L S164G L166V L222V R242E N249E L253I 20 95 10 949 437 Y11V K19T D59N I96L S164G L166I L222V R242E N249E G251D 20 114 22 520 438 Y11I K19T I96L S164G L166V R242E N249E G251D 30 93 7 1262 439 Y11V K19T D59N I96L S164G L166V L222V R242E G251D 25 108 26 423 440 Y11V K19T D59N I96L S164G L222V R242E N249E G251D 26 105 9 1196 441 Y11I K19T L222V R242E N249E G251D 46 122 26 469 442 Y11V K19T I96L L222V R242E N249E G251D 47 105 21 503 443 Y11I K19T D59N I96L S164G L166V L222V R242E N249E G251D 18 105 18 595 444 Y11V K19T I96L S164G L166V L222V R242E N249E G251D 22 96 8 1242 445 Y11I K19T D59N I96L S164G L166I L222V R242E N249E G251D 19 82 12 707 446 Y11I I96L S164G L166V L222V R242E N249E G251D 21 95 16 579 447 Y11I K19T D59N I96L S164G L222V R242E N249E 23 90 11 790 448 Y11I K19T D59N I96L L222V R242E N249E G251D 33 153 40 381 449 Y11I K19T D59N I96L S164G L222I R242E 18 89 16 564 450 Y11I K19T D59N I96L S164G L166V R242E G251D 27 88 5 1686 451 Y11I K19T D59N S164G L166I L222V R242E G251D 27 93 21 440 452 Y11I I96L L222V R242E N249E G251D 52 122 22 566 453 Y11I I96L S164G L222I R242E 15 74 5 1375 454 Y11V K19T I96L L166V L222V R242E G251D 38 119 52 228 455 Y11I D59N I96L S164G L222I R242E G251D 17 105 9 1139 456 Y11I D59N I96L S164G L222V R242E N249E G251D 23 95 15 615 457 Y11I K19T D59N I96L S164G L222I R242E N249E G251D 20 101 7 1419 458 Y11I D59N I96L S164G L166V L222V R242E G251D 22 89 16 572 459 Y11V K19T D59N I96L L222V R242E G251D 42 143 62 230 460 Y11I K19T S164G L166I L222V R242E N249E G251D 23 80 13 625 461 Y11I D59N I96L S164G L166V L222V R242E N249E G251D 20 96 35 273

In Table 15 are shown camel chymosin variants with data on cleavage of β-casein 192/193. All 45 variants show reduced proteolytic activity compared to wild type camel chymosin.

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The invention claimed is:
 1. An isolated chymosin polypeptide variant, wherein: (a) the isolated chymosin polypeptide variant has a specific clotting activity (IMCU/mg total protein) that is at least 70% of the specific clotting activity of isolated camel chymosin polypeptide having SEQ ID NO:4; (b) the isolated chymosin polypeptide variant cleaves β-casein with a frequency of less than 50% of the frequency of β-casein cleavage by the isolated camel chymosin polypeptide having SEQ ID NO:4, as determined by quantifying β-casein peptides obtained by incubating skim milk with the chymosin variant or the camel chymosin, wherein quantification is carried out by RP-HPLC coupled to an ESI-Q-TOF mass spectrometer; (c) the isolated chymosin polypeptide variant comprises an amino acid sequence comprising from 3 to fewer than 30 amino acid alterations as compared to the amino acid sequence of SEQ ID NO: 4; and (d) the isolated chymosin polypeptide variant comprises an L222V substitution relative to SEQ ID NO:4.
 2. The isolated chymosin polypeptide variant of claim 1, wherein the polypeptide variant has at least 75% of the specific clotting activity of the isolated camel chymosin polypeptide having SEQ ID NO:4.
 3. The isolated chymosin polypeptide variant of claim 1, wherein the polypeptide variant exhibits unspecific proteolytic activity (P) that is less than 50% that of the isolated camel chymosin polypeptide having SEQ ID NO:4.
 4. The isolated chymosin polypeptide variant of claim 1, wherein the polypeptide variant has a clotting activity to proteolytic activity (C/P) ratio of at least 300% of the C/P ratio of the isolated camel chymosin polypeptide having SEQ ID NO:4.
 5. The isolated chymosin polypeptide variant of claim 1, wherein, in addition to the L222V substitution, the variant further comprises one or more amino acid substitutions at a position relative to SEQ ID NO:4 selected from: L130, S132, V32, S226, V221, S255, S277, L253, M157, S271, H76, V183, I263, T239, Y307, R67, G251, R61, E83, D59, S273, Y21, V203, E294, G289, T284, Y127, V248, K321, V205, R316, D158, L166, R242 and I96.
 6. The isolated chymosin polypeptide variant of claim 1, wherein, in addition to the L222V substitution, the variant further comprises one or more amino acid substitutions relative to SEQ ID NO:4 selected from: R266V, L12M, V260T, V51L, Q288E, E83S, G251W, S154A, L180I, L215V, D144Q, I303L, L105E, K321P, E262T, and R242E.
 7. A method for making an isolated chymosin polypeptide variant according to claim 1, comprising: (a) making an alteration at from 3 to fewer than 30 positions in a DNA sequence encoding the polypeptide of SEQ ID NO:4 to obtain a DNA sequence encoding the chymosin polypeptide variant, wherein the variant comprises a L222V substitution relative to SEQ ID NO:4; and (b) producing and isolating the chymosin polypeptide variant.
 8. The method of claim 7, wherein, in addition to the L222V substitution, the polypeptide variant further comprises one or more of the following substitutions relative to SEQ ID NO:4: R266V, L12M, V260T, V51L, Q288E, E83S, G251W, S154A, L180I, L215V, D144Q, I303L, L105E, K321P, E262T, and R242E.
 9. A method for making a food or feed product comprising adding an effective amount of the isolated chymosin polypeptide variant according to claim 1 to the food or feed ingredient(s) and carrying our further manufacturing steps to obtain the food or feed product.
 10. The method according to claim 9, wherein the food or feed product is a milk-based product.
 11. A food or feed product comprising a chymosin polypeptide variant according to claim
 1. 12. The method of claim 9, wherein the food or feed product is a cheese.
 13. The method of claim 9, wherein the food or feed product is selected from pasta filata, cheddar cheese, continental type cheeses, soft cheese or white brine cheese.
 14. The method of claim 12, wherein the method is effective to reduce bitterness in the cheese.
 15. The isolated chymosin polypeptide variant of claim 1, wherein, in addition to the L222V substitution, the variant further comprises one or more amino acid substitutions relative to SEQ ID NO:4 selected from: L130I, S132A, V32L, S226T, V221M, S255Y, S277N, L253I, M157L, S271P, H76Q, V183I, I263L, T239S, Y307F, R67Q, G251D, R61Q, D59N, S273Y, Y21S, V203A, E294Q, G289S, T284S, Y127F, V248I, V205I, K231N, R316L, M256L, D158S, L166V, and I96L.
 16. The isolated chymosin polypeptide variant of claim 1, wherein, in addition to the L222V substitution, the variant further comprises an amino acid substitution at position S164 relative to SEQ ID NO:4.
 17. The isolated chymosin polypeptide variant of claim 1, wherein, in addition to the L222V substitution, the variant further comprises the amino acid substitution S164G relative to SEQ ID NO:4.
 18. The isolated chymosin polypeptide variant of claim 1, wherein the variant comprises substitutions relative to SEQ ID NO:4 selected from: K19T+D59N+I96L+S164G+L222V+N249E+G251D+I263V; Y11I+K19T+196L+S164G+L222V+R242E+G251D; K19 T+D59N+I96L+S164G+L222V+R242E+N249D+L253I; Y11V+K19T+D59N+I96L+L166V+L222V+R242E+N249E+G251D+L253I; Y11V+K19T+D59N+I96L+S164G+L222V+R242E+G251D; Y11V+K19 T+D59N+I96L+S164G+L166V+L222V+R242E+N249E+G251D; Y11V+K19 T+D59N+I96L+S164G+L166I+L222V+R242E+N249E+G251D+L253I; Y11V+D59N+I96L+S164G+L166I+L222V+R242E+G251D+L253I; Y11I+D59N+I96L+S164G+L166V+L222V+R242E+G251D+L253I; Y11V+K19T+D59N+I96L+S164G+L166I+L222V+R242E+G251D; Y11I+K19T+D59N+I96L+S164G+L166V+L222V+R242E+N249E+L253I; Y11V+K19T+D59N+I96L+S164G+L166I+L222V+R242E+N249E+G251D; Y11V+K19T+D59N+I96L+S164G+L166V+L222V+R242E+G251D; Y11V+K19T+D59N+I96L+S164G+L222V+R242E+N249E+G251D; Y11I+K19T+L222V+R242E+N249E+G251D; Y11V+K19T+196L+L222V+R242E+N249E+G251D; Y11I+K19T+D59N+I96L+S164G+L166V+L222V+R242E+N249E+G251D; Y11V+K19T+196L+S164G+L166V+L222V+R242E+N249E+G251D; Y11I+K19T+D59N+I96L+S164G+L166I+L222V+R242E+N249E+G251D; Y11I+196 L+S164G+L166V+L222V+R242E+N249E+G251D; Y11I+K19T+D59N+I96L+S164G+L222V+R242E+N249E; Y11I+K19T+D59N+I96L+L222V+R242E+N249E+G251D; Y11I+K19T+D59N+S164G+L166I+L222V+R242E+G251D; Y11V+K19T+196L+L166V+L222V+R242E+G251D; Y11I+D59N+I96L+S164G+L222V+R242E+N249E+G251D; Y11I+D59N+I96L+S164G+L166V+L222V+R242E+G251D; Y11V+K19T+D59N+I96L+L222V+R242E+G251D; Y11I+K19T+S164G+L166I+L222V+R242E+N249E+G251D, and Y11I+D59N+I96L+S164G+L166V+L222V+R242E+N249E+G251D.
 19. The isolated chymosin polypeptide variant of claim 1, wherein the variant comprises substitutions relative to SEQ ID NO:4 selected from: Y11V+K19T+D59N+I96L+S164G+L222V+R242E+G251D; Y11V+K19T+D59N+I96L+S164G+L222V+R242E+N249E+G251D; Y11V+K19T+196L+S164G+L166V+L222V+R242E+N249E+G251D; and Y11I+K19T+D59N+I96L+S164G+L222V+R242E+N249E.
 20. A method for making a food or feed product, comprising adding an effective amount of the isolated chymosin polypeptide variant according to claim 18 to the food or feed ingredient(s).
 21. The method according to claim 20, wherein the food or feed product is a cheese.
 22. A food or feed product comprising a chymosin polypeptide variant according to claim
 18. 23. The food or feed product according to claim 22, wherein the food or feed product is a cheese.
 24. A method for making an isolated chymosin polypeptide variant according to claim 18, comprising: (a) making alterations in a DNA sequence encoding the polypeptide of SEQ ID NO:4 to obtain a DNA sequence encoding the chymosin polypeptide variant, and (b) producing and isolating the chymosin polypeptide variant. 